Heat shock protein-based vaccines and immunotherapies

ABSTRACT

The present invention relates to methods and compositions for inducing an immune response in a subject, wherein the subject is administered an effective amount of at least one heat shock protein in combination with one or more defined target antigens. These methods and compositions may be used in the treatment of infectious diseases and cancers.

[0001] The invention described herein was made in the course of workunder NIH Core Grant No. CA 08748. The United States government may havecertain rights in this invention.

INTRODUCTION

[0002] The present invention relates to methods and compositions forinducing an immune response in a subject, wherein the subject isadministered an effective amount of at least one heat shock protein incombination with one or more defined target antigens. These methods andcompositions may be used in the treatment of infectious diseases andcancers.

BACKGROUND OF THE INVENTION

[0003] Heat shock proteins were originally observed to be expressed inincreased amounts in mammalian cells which were exposed to suddenelevations of temperature, while the expression of most cellularproteins is significantly reduced. It has since been determined thatsuch proteins are produced in response to various types of stress,including glucose deprivation. As used herein, the term “heat shockprotein” will be used to encompass both proteins that are expresslylabeled as such as well as other stress proteins, including homologs ofsuch proteins that are expressed constitutively (i.e., in the absence ofstressful conditions). Examples of heat shock proteins include BiP (alsoreferred to as grp78), hsp/hsc70, gp96 (grp94), hsp60, hsp40 and hsp90.

[0004] Heat shock proteins have the ability to bind other proteins intheir non-native states, and in particular to bind nascent peptidesemerging from ribosomes or extruded into the endoplasmic reticulum.Hendrick and Hartl, Ann. Rev. Biochem. 62:349-384 (1993); Hartl, Nature381:571-580 (1996). Further, heat shock proteins have been shown to playan important role in the proper folding and assembly of proteins in thecytosol, endoplasmic reticulum and mitochondria; in view of thisfunction, they are referred to as “molecular chaperones.” Frydman etal., Nature 370:111-117 (1994); Hendrick and Hartl, Ann. Rev. Biochem.62:349-384 (1993); Hartl, Nature 381:571-580 (1996).

[0005] For example, the protein BiP, a member of a class of heat shockproteins referred to as the hsp70 family, has been found to bind tonewly synthesized, unfolded μ immunoglobulin heavy chain prior to itsassembly with light chain in the endoplasmic reticulum. Hendershot etal., J. Cell Biol. 104:761-767 (1987). Another heat shock protein, gp96,is a member of the hsp90 family of stress proteins which localizes inthe endoplasmic reticulum. Li and Srivastava, EMBO J. 12:3143-3151(1993); Mazzarella and Green, J. Biol. Chem. 262:8875-8883 (1987). Ithas been proposed that gp96 may assist in the assembly of multi-subunitproteins in the endoplasmic reticulum. Wiech et al., Nature 358:169-170(1992).

[0006] It has been observed that heat shock proteins prepared fromtumors in experimental animals were able to induce immune responses in atumor-specific manner; that is to say, heat shock protein purified froma particular tumor could induce an immune response in an experimentalanimal which would inhibit the growth of the same tumor, but not othertumors. Srivastava and Maki, Curr. Topics Microbiol. 167:109-123 (1991).The source of the tumor-specific immunogenicity has not been confirmed.Genes encoding heat shock proteins have not been found to exhibittumor-specific DNA polymorphism. Srivastava and Udono, Curr. Opin.Immunol. 6:728-732 (1994). High resolution gel electrophoresis hasindicated that gp96 may be heterogeneous at the molecular level. Feldwegand Srivastava, Int. J. Cancer 63:310-314 (1995). Evidence suggests thatthe source of heterogeneity may be populations of small peptidesadherent to the heat shock protein, which may number in the hundreds.Id. It has been proposed that a wide diversity of peptides adherent totumor-synthesized heat shock proteins may render such proteins capableof eliciting an immune response in subjects having diverse HLAphenotypes, in contrast to more traditional immunogens which may besomewhat HLA-restricted in their efficacy. Id.

[0007] Recently, Nieland et al. (Proc. Natl. Acad. Sci. U.S.A.93:6135-6139 (1996)) identified an antigenic peptide containing acytotoxic T lymphocyte (CTL) vesicular stomatitis virus (VSV) epitopebound to gp96 produced by VSV-infected cells. Neiland's methodsprecluded the identification of any additional peptides or othercompounds which may also have bound to gp96, and were therefore unableto further characterize higher molecular weight material which was boundto gp96 and detected by high pressure liquid chromatography.

[0008] It has been reported that a synthetic peptide comprising multipleiterations of NANP (Asp Ala Asp Pro) malarial antigen, chemicallycross-linked to glutaraldehyde-fixed mycobacterial hsp65 or hsp70, wascapable of inducing antibody formation (i.e., a humoral response) inmice in the absence of any added adjuvant; a similar effect was observedusing heat shock protein from the bacterium Escherichia coli. DelGuidice, Experientia 50:1061-1066 (1994); Barrios et al., Clin. Exp.Immunol. 98:224-228 (1994); Barrios et al., Eur. J. Immunol.22:1365-1372 (1992). Cross-linking of synthetic peptide to heat shockprotein and possibly glutaraldehyde fixation was required for antibodyinduction. Barrios et al., Clin. Exp. Immunol. 98:229-233.

[0009] It has now been discovered, according to the present invention,that heat shock protein may be combined with target antigen and used toinduce an immune response which includes a cytotoxic cellular component,i.e., a cellular response.

SUMMARY OF THE INVENTION

[0010] The present invention relates to methods and compositions forinducing an immune response in a subject, wherein at least one heatshock protein in combination with one or more defined target antigens isadministered to the subject.

[0011] Unlike prior disclosures relating to heat shock proteinassociated with an undefined population of potential antigens which havebeen restricted, in their immunogenic effect, to a single tumor, thepresent invention provides for methods and compositions which combineheat shock protein with a defined target antigen which may be selectedon the basis that it is immunogenic in diverse occurrences of aneoplastic or infectious disease, or because it has been identified, inan individual instance, as being particularly immunogenic. Further,because the use of one or more defined target antigen permits morecontrol over the immune response elicited, it may avoid the induction ofan undesirable immune response.

[0012] In alternative embodiments of the invention, the target antigenmay be either (i) an antigen which itself binds to the heat shockprotein; or (ii) a hybrid antigen comprising an immunogenic domain aswell as a heat shock protein-binding domain. The immunogenic domain maybe an entire protein or peptide antigen, or may be only a portion of theselected antigen, for example a selected epitope of the antigen. Inspecific, nonlimiting embodiments of the invention, the heat shockprotein binding domain may comprise a peptide having the sequence:

[0013] His Trp Asp Phe Ala Trp Pro Trp (SEQ ID NO:1).

[0014] The present invention provides for methods of administering suchheat shock protein/target antigen compositions comprising (i) combiningone or more heat shock protein with one or more target antigens invitro, under conditions wherein binding of target antigen to heat shockprotein occurs to form a target antigen/heat shock protein complex; and(ii) administering the target antigen, bound to heat shock protein, inan effective amount to a subject in need of such treatment.

[0015] Alternatively, heat shock protein/target antigen combinations ofthe invention may be administered to a subject by introducing nucleicacid encoding the heat shock protein and the target antigen into thesubject such that the heat shock protein and target antigen bind insitu.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the induction of a cellular immune response usinghybrid peptide antigens in accordance with the invention.

[0017]FIG. 2 shows the induction of a cellular immune response usinghybrid peptide antigens in accordance with the invention.

[0018]FIG. 3 shows the induction of a cellular immune response usinghybrid peptide antigens in accordance with the invention.

[0019]FIG. 4 shows the induction of a cellular immune response usinghybrid peptide antigens in accordance with the invention.

[0020]FIGS. 5A and 5B shows the results of control experiments in whichhybrid peptide or Ova-peptide and heat shock protein were administeredindividually to EL4 cells.

[0021]FIG. 6 shows co-elution of hybrid peptides and heat shock proteinsfrom a column, demonstrating binding of the polypeptides to the heatshock protein.

[0022]FIG. 7 shows the co-elution of ¹²⁵I-OVA-BiP with BiP in thepresence and absence of ATP.

[0023]FIG. 8 shows the killing efficacy of T-cells primed with variouscombinations of antigens and heat shock proteins on EL4 cells pulsedwith antigen.

[0024]FIG. 9 shows the killing efficacy of T-cells primed with variousconcentrations of antigens and heat shock proteins on EG7 lymphomacells.

DETAILED DESCRIPTION OF THE INVENTION

[0025] For purposes of clarity of description, and not by way oflimitation, the detailed description is divided into the followingsubsections:

[0026] (i) heat shock proteins;

[0027] (ii) target antigens; and

[0028] (iii) methods of administration.

[0029] Heat Shock Proteins

[0030] The term “heat shock protein,” as used herein, refers to anyprotein which exhibits increased expression in a cell when the cell issubjected to a stress. In preferred nonlimiting embodiments, the heatshock protein is originally derived from a eukaryotic cell; in morepreferred embodiments, the heat shock protein is originally derived froma mammalian cell. For example, but not by way of limitation, heat shockproteins which may be used according to the invention include BiP (alsoreferred to as grp78), hsp/hsc70, gp96 (grp94), hsp60, hsp40, and hsp90.Especially preferred heat shock proteins are BiP, gp96, and hsp70, asexemplified below. Naturally occurring or recombinantly derived mutantsof heat shock proteins may also be used according to the invention. Forexample, but not by way of limitation, the present invention providesfor the use of heat shock proteins mutated so as to facilitate theirsecretion from the cell (for example having mutation or deletion of anelement which facilitates endoplasmic reticulum recapture, such as KDELor its homologs; such mutants are described in concurrently filed PCTApplication No. PCT/US96/13233 (WO 97/06685), which is incorporatedherein by reference).

[0031] For embodiments of the invention wherein heat shock protein andtarget antigen are directly administered to the subject in the form of aprotein/peptide complex, the heat shock protein may be prepared, usingstandard techniques, from natural sources, for example as described inFlynn et al., Science 245:385-390 (1989), or using recombinanttechniques such as expression of a heat shock encoding vector in asuitable host cell such as a bacterial, yeast or mammalian cell. Ifpre-loading of the heat shock protein with peptides from the hostorganism is a concern, the heat shock protein can be incubated with ATPand then repurified. Nonlimiting examples of methods for preparingrecombinant heat shock proteins are set forth below.

[0032] A nucleic acid encoding a heat shock protein may be operativelylinked to elements necessary or desirable for expression and then usedto express the desired heat shock protein as either a means to produceheat shock protein for use in a protein vaccine or, alternatively, in anucleic acid vaccine. Elements necessary or desirable for expressioninclude, but are not limited to, promoter/enhancer elements,transcriptional start and stop sequences, polyadenylation signals,translational start and stop sequences, ribosome binding sites, signalsequences and the like. For example, but not by way of limitation, genesfor various heat shock proteins have been cloned and sequenced,including, but not limited to, gp96 (human: Genebank Accession No.X15187; Maki et al., Proc. Natl. Acad. Sci. U.S.A. 87:5658-5562 (1990);mouse: Genebank Accession No. M16370; Srivastava et al., Proc. Natl.Acad. Sci. U.S.A. 84:3807-3811 (1987)), BiP (mouse: Genebank AccessionNo. U16277; Haas et al., Proc. Natl. Acad. Sci. U.S.A. 85:2250-2254(1988); human: Genebank Accession No. M19645; Ting et al., DNA 7:275-286(1988)), hsp70 (mouse: Genebank Accession No. M35021; Hunt et al., Gene87:199-204 (1990); human: Genebank Accession No. M24743; Hunt et al,Proc. Natl. Acad. Sci. U.S.A. 82:6455-6489 (1995)), and hsp40 (human:Genebank Accession No. D49547; Ohtsuka K., Biochem. Biophys. Res.Commun. 197:235-240 (1993)).

[0033] Target Antigens

[0034] A target antigen, according to the invention, may be either (i)an antigen which itself binds to the heat shock protein; or (ii) ahybrid antigen comprising an immunogenic domain as well as a heat shockprotein-binding domain. Thus, the target antigen serves at least twofunctions, namely (i) it contains an epitope capable of inducing thedesired immune response; and (ii) it is capable of physically binding toits partner heat shock protein. Of note, the term “physically binds”indicates that the target antigen and heat shock protein exhibit aphysical interaction which permits the adherence of one to the other forat least a transient period of time; of note, the binding need not, andin most embodiments of the invention should not, be irreversible.

[0035] In certain embodiments, an antigen capable of inducing thedesired immune response may be found to be inherently capable of bindingto a partner heat shock protein. In other embodiments, it may benecessary or desirable to link an immunogenic antigen to one or moreother compounds so as to create a hybrid antigen which contains both animmunogenic domain as well as a heat shock protein binding domain. Insuch circumstances, a compound which is, itself, an immunogenic antigenmay be linked to a compound which is, itself, capable of binding to aheat shock protein. Alternatively, the linkage of two or more compoundswhich individually lack either functionality may give rise to thedesired immunogenic and binding characteristics.

[0036] The term “antigen” as used herein, refers to a compound which maybe composed of amino acids, carbohydrates, nucleic acids or lipidsindividually or in any combination.

[0037] The term “target antigen,” as used herein, refers to a compoundwhich binds to one or more heat shock proteins and which isrepresentative of the immunogen toward which an immune response isdesirably directed. For example, where the immunogen is an influenzavirus, the target antigen may be a peptide fragment of the matrixprotein of the influenza virus. As used herein, the term “immunogen” isapplied to the neoplastic cell, infected cell, pathogen, or componentthereof, towards which an immune response is to be elicited, whereas thetarget antigen is a portion of that immunogen which can provoke thedesired response and which inherently or through engineering binds toone or more heat shock proteins. In particular, the target antigen isselected to elicit an immune response to a particular disease orpathogen, including peptides obtained from MHC molecules, mutated DNAgene products, and direct DNA products such as those obtained from tumorcells.

[0038] While the invention may be applied to any type of immunogen,immunogens of particular interest are those associated with, derivedfrom, or predicted to be associated with a neoplastic disease, includingbut not limited to a sarcoma, a lymphoma, a leukemia, or a carcinoma,and in particular, with melanoma, carcinoma of the breast, carcinoma ofthe prostate, ovarian carcinoma, carcinoma of the cervix, coloncarcinoma, carcinoma of the lung, glioblastoma, astrocytoma, etc.Further, mutations of tumor suppressor gene products such as p53, oroncogene products such as ras may also provide target antigens to beused according to the invention.

[0039] In further embodiments, the immunogen may be associated with aninfectious disease, and, as such, may be a bacterium, virus, protozoan,mycoplasma, fungus, yeast, parasite, or prion. For example, but not byway of limitation, the immunogen may be a human papilloma virus (seebelow), a herpes virus such as herpes simplex or herpes zoster, aretrovirus such as human immunodeficiency virus 1 or 2, a hepatitisvirus, an influenza virus, a rhinovirus, respiratory syncytial virus,cytomegalovirus, adenovirus, Mycoplasma pneumoniae, a bacterium of thegenus Salmonella, Staphylococcus, Streptococcus, Enterococcus,Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, amalarial parasite, Trypanosoma cruzi, etc.

[0040] Immunogens may be obtained by isolation directly from a neoplasm,an infected cell, a specimen from an infected subject, a cell culture,or an organism culture, or may be synthesized by chemical or recombinanttechniques. Suitable antigenic peptides, particularly for use in ahybrid antigen, for use against viruses, bacteria and the like can bedesigned by searching through their sequences for MHC class I restrictedpeptide epitopes containing HLA binding sequences such as but notlimited to HLA-A2 peptide binding sequences:Xaa(Leu/Met)XaaXaaXaa(Val/Ile/Leu/Thr)XaaXaa(Val/Leu) (SEQ ID NO:2), forexample, from viruses: Ser Gly Pro Ser Asn Thr Pro Pro Glu Ile; (SEQ IDNO:31) Ser Gly Val Glu Asn Pro Gly Gly Tyr Cys Leu; (SEQ ID NO:32) LysAla Val Tyr Asn Phe Ala Thr Cys Gly; (SEQ ID NO:33) Arg Pro Gln Ala SerGly Val Tyr Met; (SEQ ID NO:34) Phe Gln Pro Gln Asn Gly Gln Phe Ile;(SEQ ID NO:35) Ile Glu Gly Gly Trp Thr Gly Met Ile; (SEQ ID NO:36) ThrTyr Val Ser Val Ser Thr Ser Thr Leu; (SEQ ID NO:37) Phe Glu Ala Asn GlyAsn Leu Ile; (SEQ ID NO:38) Ile Tyr Ser Thr Val Ala Ser Ser Leu; (SEQ IDNO:39) Thr Tyr Gln Arg Thr Arg Ala Leu Val; (SEQ ID NO:40) Cys Thr GluLeu Lys Leu Ser Asp Tyr; (SEQ ID NO:41) Ser Asp Tyr Glu Gly Arg Leu Ile;(SEQ ID NO:42) Glu Glu Gly Ala Ile Val Gly Glu Ile; (SEQ ID NO:43) ValSer Asp Gly Gly Pro Asn Leu Tyr; (SEQ ID NO:44) Ala Ser Asn Glu Asn MetGlu Thr Met; (SEQ ID NO:45) Ala Ser Asn Glu Asn Met Asp Ala Met; (SEQ IDNO:46) Lys Leu Gly Glu Phe Tyr Asn Gln Met Met; (SEQ ID NO:47) Leu TyrGln Asn Val Gly Thr Tyr Val; (SEQ ID NO:48) Thr Tyr Val Ser Val Gly ThrSer Thr Leu; (SEQ ID NO:49) Phe Glu Ser Thr Gly Asn Leu Ile; (SEQ IDNO:50) Val Tyr Gln Ile Leu Ala Ile Tyr Ala; (SEQ ID NO:51) Ile Tyr AlaThr Val Ala Gly Ser Leu; (SEQ ID NO:52) Gly Ile Leu Gly Phe Val Phe ThrLeu; (SEQ ID NO:53) Ile Leu Gly Phe Val Phe Thr Leu Thr Val; (SEQ IDNO:54) Ile Leu Arg Gly Ser Val Ala His Lys; (SEQ ID NO:55) Glu Asp LeuArg Val Leu Ser Phe Ile; (SEQ ID NO:56) Glu Leu Arg Ser Arg Tyr Trp AlaIle; (SEQ ID NO:57) Ser Arg Tyr Trp Ala Ile Arg Thr Arg; (SEQ ID NO:58)Lys Thr Gly Gly Pro Ile Tyr Lys Arg; (SEQ ID NO:59) Phe Ala Pro Gly AsnTyr Pro Ala Leu; (SEQ ID NO:60) Arg Arg Tyr Pro Asp Ala Val Tyr Leu;(SEQ ID NO:61) Asp Pro Val Ile Asp Arg Leu Tyr Leu; (SEQ ID NO:62) SerPro Gly Arg Ser Phe Ser Tyr Phe; (SEQ ID NO:63) Tyr Pro Ala Leu Gly LeuHis Glu Phe; (SEQ ID NO:64) Thr Tyr Lys Asp Thr Val Gln Leu; (SEQ IDNO:65) Phe Tyr Asp Gly Phe Ser Lys Val Pro Leu; (SEQ ID NO:66) Phe IleAla Gly Asn Ser Ala Tyr Glu Tyr Val; (SEQ ID NO:67) Tyr Pro His Phe MetPro Thr Asn Leu; (SEQ ID NO:68) Ala Pro Thr Ala Gly Ala Phe Phe Phe;(SEQ ID NO:69) Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg; (SEQ IDNO:70) Phe Leu Pro Ser Asp Phe Phe Pro Ser Val; (SEQ ID NO:71) Trp LeuSer Leu Leu Val Pro Phe Val; (SEQ ID NO:72) Gly Leu Ser Pro Thr Val TrpLeu Ser Val; (SEQ ID NO:73) Asp Leu Met Gly Tyr Ile Pro Leu Val; (SEQ IDNO:74) Leu Met Gly Tyr Ile Pro Leu Val Gly Ala; (SEQ ID NO:75) Ala SerArg Cys Trp Val Ala Met; (SEQ ID NO:76) Lys Leu Val Ala Leu Gly Ile AsnAla Val; (SEQ ID NO:77) Phe Leu Arg Gly Arg Ala Tyr Gly Leu; (SEQ IDNO:78) Arg Arg Ile Tyr Asp Leu Ile Glu Leu; (SEQ ID NO:79) Ile Val ThrAsp Phe Ser Val Ile Lys; (SEQ ID NO:80) Arg Arg Arg Trp Arg Arg Leu ThrVal; (SEQ ID NO:81) Glu Glu Asn Leu Leu Asp Phe Val Arg Phe; (SEQ IDNO:82) Cys Leu Gly Gly Leu Leu Thr Met Val; (SEQ ID NO:83) Ser Ser IleGlu Phe Ala Arg Leu; (SEQ ID NO:84) Leu Tyr Arg Thr Phe Ala Gly Asn ProArg Ala; (SEQ ID NO:85) Asp Tyr Ala Thr Leu Gly Val Gly Val; (SEQ IDNO:86) Leu Leu Leu Gly Thr Leu Asn Ile Val; (SEQ ID NO:87) Leu Leu MetGly Thr Leu Gly Ile Val; (SEQ ID NO:88) Thr Leu Gln Asp Ile Val Leu HisLeu; (SEQ ID NO:89) Gly Leu His Cys Tyr Glu Gln Leu Val; (SEQ ID NO:90)Pro Leu Lys Gln His Phe Gln Ile Val; (SEQ ID NO:91) Arg Leu Val Thr LeuLys Asp Ile Val; (SEQ ID NO:92) Arg Ala His Tyr Asn Ile Val Thr Phe;(SEQ ID NO:93) Leu Leu Phe Gly Tyr Pro Val Tyr Val; (SEQ ID NO:94) SerAla Ile Asn Asn Tyr Ala Gln Lys Leu; (SEQ ID NO:95) His Gln Ala Ile SerPro Arg Thr Leu; (SEQ ID NO:96) Gln Met Val His Gln Ala Ile Ser Pro ArgThr Leu; (SEQ ID NO:97) Cys Lys Gly Val Asn Lys Glu Tyr Leu; (SEQ IDNO:98) Gln Gly Ile Asn Asn Leu Asp Asn Leu; (SEQ ID NO:99) Asn Asn LeuAsp Asn Leu Arg Asp Tyr; (SEQ ID NO:100) Ser Glu Phe Leu Leu Glu Lys ArgIle; (SEQ ID NO:101) Ser Tyr Ile Gly Ser Ile Asn Asn Ile; (SEQ IDNO:102) Ile Leu Gly Asn Lys Ile Val Arg Met Tyr; (SEQ ID NO:103) Arg LeuArg Pro Gly Gly Lys Lys Lys; (SEQ ID NO:104) Glu Ile Lys Asp Thr Lys GluAla Leu; (SEQ ID NO:105) Gly Glu Ile Tyr Lys Arg Trp Ile Ile; (SEQ IDNO:106) Glu Ile Tyr Lys Arg Trp Ile Ile Leu; (SEQ ID NO:107) Arg Tyr LeuLys Asp Gln Gln Leu Leu; (SEQ ID NO:108) Arg Gly Pro Gly Arg Ala Phe ValThr Ile; (SEQ ID NO:109) Ile Val Gly Leu Asn Lys Ile Val Arg; (SEQ IDNO:110) Thr Val Tyr Tyr Gly Val Pro Val Trp Lys; (SEQ ID NO:111) Arg LeuArg Asp Leu Leu Leu Ile Val Thr Arg; (SEQ ID NO:112) Lys Arg Trp Ile IleLeu Gly Leu Asn Lys; (SEQ ID NO:113) Ser Phe Asn Cys Gly Gly Glu PhePhe; (SEQ ID NO:114) Gly Arg Ala Phe Val Thr Ile Gly Lys; (SEQ IDNO:115) Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu; (SEQ ID NO:116) Gln ValPro Leu Arg Pro Met Thr Tyr Lys; (SEQ ID NO:117) Thr Glu Met Glu Lys GluGly Lys Ile; (SEQ ID NO:118) Ile Leu Lys Glu Pro Val His Gly Val; (SEQID NO:119) Val Glu Ala Glu Ile Ala His Gln Ile; (SEQ ID NO:120) Arg GlyTyr Val Tyr Gln Gly Leu; (SEQ ID NO:121) Tyr Ser Gly Tyr Ile Phe Arg AspLeu; (SEQ ID NO:122) Val Gly Pro Val Phe Pro Pro Gly Met; (SEQ IDNO:123) Ile Ile Tyr Arg Phe Leu Leu Ile; (SEQ ID NO:124)

[0041] (SEQ ID NO:125) Lys Tyr Gly Val Ser Val Gln Asp Ile; (SEQ IDNO:126) Ile Gln Val Gly Asn Thr Arg Thr Ile; (SEQ ID NO:127) Thr Pro HisPro Ala Arg Ile Gly Leu;

[0042] (SEQ ID NO:128) Ser Tyr Ile Pro Ser Ala Glu Lys Ile; (SEQ IDNO:129) Lys Pro Lys Asp Glu Leu Asp Tyr; (SEQ ID NO:130) Lys Ser Lys AspGlu Leu Asp Tyr; (SEQ ID NO:131) Lys Pro Asn Asp Lys Ser Leu Tyr; (SEQID NO:132) Lys Tyr Leu Lys Lys Ile Lys Asn Ser Leu; (SEQ ID NO:133) TyrGlu Asn Asp Ile Glu Lys Lys Ile; (SEQ ID NO:134) Asn Tyr Asp Asn Ala GlyThr Asn Leu; (SEQ ID NO:135) Asp Glu Leu Asp Tyr Glu Asn Asp Ile; (SEQID NO:136) Ser Tyr Val Pro Ser Ala Glu Gln Ile;

[0043] (SEQ ID NO:137) Phe Glu Gln Asn Thr Ala Gln Pro; (SEQ ID NO:138)Phe Glu Gln Asn Thr Ala Gln Ala; (SEQ ID NO:139) Glu Ala Asp Pro Thr GlyHis Ser Tyr; (SEQ ID NO:140) Glu Val Asp Pro Ile Gly His Leu Tyr; (SEQID NO:141) Ala Ala Gly Ile Gly Ile Leu Thr Val; (SEQ ID NO:142) Tyr LeuGlu Pro Gly Pro Val Thr Ala; (SEQ ID NO:143) Ile Leu Asp Gly Thr Ala ThrLeu Arg Leu; (SEQ ID NO:144) Met Leu Leu Ala Leu Leu Tyr Cys Leu; (SEQID NO:145) Tyr Met Asn Gly Thr Met Ser Gln Val; (SEQ ID NO:146) Leu ProTyr Leu Gly Trp Leu Val Phe; (SEQ ID NO:147) Phe Gly Pro Tyr Lys Leu AsnArg Leu; (SEQ ID NO:148) Lys Ser Pro Trp Phe Thr Thr Leu; (SEQ IDNO:149) Gly Pro Pro His Ser Asn Asn Phe Gly Tyr; and (SEQ ID NO:150) IleSer Thr Gln Asn His Arg Ala Leu; (SEQ ID NO:3) (Rammensee et al.,Immunogenetics 41:178-223 (1995)), Xaa(Leu/Met)XaaXaaXaaXaaXaaXaaVal(SEQ ID NO:28) (Tarpey et al., Immunology 81:222-227 (1994)), Xaa(Val/Gln) XaaXaaXaaXaaXaaXaaLeu,

[0044] for example, from virus: Tyr Gly Ile Leu Gly Lys Val Phe Thr Leu;(SEQ ID NO:151) Ser Leu Tyr Asn Thr Val Ala Thr Leu; (SEQ ID NO:152)(Barouch et al., J. Exp. Med. 182: 1347-1856 (1995)).

[0045] It may also be desirable to consider the type of immune responsewhich is desired. For example, under certain circumstances, a humoralimmune response may be appropriate. In other cases, and indeed where animmune response directed toward neoplastic cells or infected cells issought to be elicited, a cellular immune response is particularlydesirable. Accordingly, particular epitopes associated with theactivation of B cells, T helper cells, or cytotoxic T cells may beidentified and selected for incorporation into the target antigen.

[0046] It may also be desirable to utilize target antigen associatedwith an autoimmune disease or allergy. Such a target antigen may beadministered, together with one or more heat shock proteins, in anamount sufficient to be tolerogenic or to inhibit a pre-existing immuneresponse to the target antigen in a subject. The amount of heat shockprotein required to inhibit the immune response is expected to besubstantially greater than the amount required for stimulation.

[0047] Although the size of target antigen may vary depending upon theheat shock protein used, in nonlimiting embodiments of the invention,the target antigen may be the size of a peptide having between 4 and 500amino acid residues, and preferably be the size of a peptide havingbetween 4 and 100, most preferably 7 and 20 amino acid residues. Assuch, it may be desirable to produce a fragment of an immunogen to serveas a target antigen, or, alternatively, to synthesize a target antigenby chemical or recombinant DNA methods. In some instances, however, animmunogen may, in intact form, serve as a target antigen.

[0048] Based on the foregoing considerations, a target antigen may beprepared, and then tested for its ability to bind to heat shock protein.In some instances, binding of target antigen to a particular heat shockprotein may be facilitated by the presence of at least one otherprotein, which may be a heat shock protein.

[0049] For example, binding of target antigen to a heat shock proteinmay be evaluated by labeling the target antigen with a detectable label,such as a radioactive, fluorescent, enzymatic or pigmented label,combining the target antigen with heat shock protein under conditionswhich would be expected to permit binding to occur, and then isolatingthe heat shock protein while removing any unbound target antigen, anddetermining whether any labeled target antigen had adhered to the heatshock protein. As a specific example, and not by way of limitation, theability of a target antigen to bind to BiP heat shock protein may beevaluated by combining 2 μg BiP with up to about 1150 pmole ofradioactively labeled target antigen in buffer containing 50 mM Tris HCl(pH 7.5), 200 mM NaCl, and 1 mM Na₂EDTA, in a final volume of 50 μl, for30 minutes at 37 degrees Centigrade. Unbound target antigen may then beremoved from bound BiP-target antigen by centrifugation at 100 g bydesalting through a 1 ml Sephadex-G column for 2 minutes. Penefsky, J.Biol. Chem. 252:2891 (1977). To prevent binding to the resin, columnsmay first be treated with 100 μl of bovine serum albumin in the samebuffer and centrifuged as above. Bound target antigen may then bequantitated by liquid scintillation counting. See Flynn et al., Science245:385-390 (1989).

[0050] Because ATP hydrolysis drives the release of peptides from manyknown heat shock proteins, the amount of ATPase activity may often beused to quantitate the amount of target antigen binding to heat shockprotein. An example of how such an assay may be performed is set forthin Flynn et al., Science 245:385-390 (1989).

[0051] If a particular immunogen or a fragment thereof does notsatisfactorily bind to a heat shock protein, then that immunogen orfragment may be linked to another compound so as to create a heat shockprotein-binding domain thereby constructing a hybrid antigen. The heatshock protein-binding domain is selected so that the hybrid peptide willbind in vitro to a heat shock protein such as BiP, hsp70, gp96, orhsp90, alone or in combination with accessory heat shock proteins suchas hsp40, or hsp60. Peptides which fulfill this criterion may beidentified by panning libraries of antigens known to bind well to one ormore heat shock proteins as described in Blond-Elguindi et al., Cell75:717-728 (1993): Leu Phe Trp Pro Phe Glu Trp Ile; (SEQ ID NO:153) AspGly Val Gly Ser Phe Ile Gly; (SEQ ID NO:154) Glu Ser Leu Trp Asn Pro GlnGly; (SEQ ID NO:155) Leu His Phe Asp Val Leu Trp Arg; (SEQ ID NO:156)Cys His Leu Lys Met Val Pro Trp; (SEQ ID NO:157) Asn Ser Val Leu Val CysGlu Leu; (SEQ ID NO:158) Asp Arg Gly His Ser Thr Tyr Ser; (SEQ IDNO:159) Asp Val Trp Gly Trp Val Thr Trp; (SEQ ID NO:160) Ile Gln Phe ArgVal Glu Leu Phe; (SEQ ID NO:161) Leu Trp Leu Glu Leu Ser Leu Ser; (SEQID NO:162) Val Gly Ile Cys Ala Leu Gly Phe; (SEQ ID NO:163) Pro Tyr ProSer Gly Leu Asp Ser; (SEQ ID NO:164) Phe Trp Gly Val Leu Pro Tyr Pro;(SEQ ID NO:165) Phe Thr His Gly Ile Ser Leu Tyr; (SEQ ID NO:166) Asn HisSer Phe Gly Gly Ser Thr; (SEQ ID NO:167) Val Asp Tyr Val Tyr Phe HisHis; (SEQ ID NO:168) Phe Leu Asp Ile Ile Gly Tyr Gly; (SEQ ID NO:169)Trp Asp Asp Leu Leu His Gly Arg; (SEQ ID NO:170) Leu Arg Leu Leu Gly ThrLeu Asn; (SEQ ID NO:171) Phe Glu Gln His Asn Gln Glu Pro; (SEQ IDNO:172) Phe Val Gly Thr Val Thr Trp Ser; (SEQ ID NO:173) Leu Trp Ala LeuThr Tyr Arg Gly; (SEQ ID NO:174) Ser Trp Gly Ser Asn Gly Gly Phe; (SEQID NO:175) Asp Met Trp Arg Arg Ala Val Gln; (SEQ ID NO:176) Cys Arg ValIle Tyr His Ala Thr; (SEQ ID NO:177) Met Val Val Ala Arg Cys Gly His;(SEQ ID NO:178) His Met Trp Ile Asn Trp Val Gln; (SEQ ID NO:179) Cys AlaGly Arg Cys Phe Gly Tyr; (SEQ ID NO:180) Cys Thr His Val Leu Ala TyrSer; (SEQ ID NO:181) Ser Trp Met Pro Trp Leu Thr Met; (SEQ ID NO:182)Leu Glu Trp Cys Ile Trp Arg Tyr; (SEQ ID NO:183) Cys Leu Ala Cys Ile IleHis Ser; (SEQ ID NO:184) Phe Trp Phe Pro Trp Asp Arg Ser; (SEQ IDNO:185) Trp Arg Thr Gly Val Phe His Gly; (SEQ ID NO:186) Met His Leu ArgVal Ala Asp Arg; (SEQ ID NO:187) Ala Leu Asp Leu Tyr Leu Tyr Val; (SEQID NO:188) Phe Phe Trp Phe Thr Leu Lys Glu; (SEQ ID NO:189) Leu Ser PheAla Gly Trp Gly Val; (SEQ ID NO:190) Met Met Met Leu Gly Arg Ala Pro;(SEQ ID NO:191) Trp Ser Phe Tyr Thr Trp Leu Asn; (SEQ ID NO:192) Phe ValTrp Met Arg Trp Ile Asp; (SEQ ID NO:193) Met Gln Val Asn Thr Pro AspAsn; (SEQ ID NO:194) Phe Trp Gly Trp Leu Ile Pro Trp; (SEQ ID NO:195)Trp Gly Trp Val Trp Trp Asp; (SEQ ID NO:196) Trp Ile Phe Pro Trp Ile GlnLeu; (SEQ ID NO:197) Trp Met Phe Asn Trp Pro Trp Tyr; (SEQ ID NO:198)Met Asn Met Ile Val Leu Asp Lys; (SEQ ID NO:199) Phe Trp Gly Trp Pro GlyTrp Ser; (SEQ ID NO:200) Trp Leu Ile Arg Val Gly Thr Ala; (SEQ IDNO:201) Gly Leu Leu Thr His Leu Ile Trp; (SEQ ID NO:202) Leu Trp Trp LeuAsn Val His Gly; (SEQ ID NO:203) Trp Trp Trp Ile Asn Asp Glu Ser; (SEQID NO:204) Ala Asn Pro Ser Leu Ala Thr Tyr; (SEQ ID NO:205) Trp Leu GlnGly Trp Trp Gly Trp; (SEQ ID NO:206) Met Met Pro Val Thr Ser Phe Arg;(SEQ ID NO:207) Gly Trp Met Asp Trp Trp Tyr Tyr; (SEQ ID NO:208) Leu AlaSer Met Arg Asn Ser Met; (SEQ ID NO:209) Asp Leu Met Arg Trp Leu GlyLeu; (SEQ ID NO:210) Tyr Phe Tyr Ala Trp Trp Leu Asp; (SEQ ID NO:211)Leu Gly His Leu Trp Thr Gln Val; (SEQ ID NO:212) Leu Trp Trp Arg Asp ValMet Ala; (SEQ ID NO:213) Phe Ile Trp Trp Ala Pro Leu Ala; (SEQ IDNO:214) Gly Ser Val Gly Gly Gly Val Val; (SEQ ID NO:215) Asp Ser His AspAsp Trp Arg Met; (SEQ ID NO:216) Phe Trp Arg Phe Asp Tyr Tyr Phe; (SEQID NO:217) Trp Thr Trp Trp Glu Trp Leu Ala; (SEQ ID NO:218) Trp Leu TrpAsp Trp Ile Val Val; (SEQ ID NO:219) Gly Trp Thr Trp Phe Phe Asp Met;(SEQ ID NO:220) Ala Trp Trp Gln His Phe Ile Val; (SEQ ID NO:221) Leu TrpTrp Asp Ile Ile Thr Gly; (SEQ ID NO:222) Phe Thr Tyr Gly Ser Arg TrpLeu; (SEQ ID NO:223) Phe Ser Leu Trp Pro Leu Ala Trp; (SEQ ID NO:224)Gly Ile Ile Leu Gly Tyr Asn Val; (SEQ ID NO:225) Ser Trp Met Thr Trp IleGlu His; (SEQ ID NO:226) Gly Trp Trp Val Thr Trp Pro Trp; (SEQ IDNO:227) Val Val Ser Pro Trp Trp Leu Gly; (SEQ ID NO:228) Asn Val Leu SerArg Gly Phe Ser; (SEQ ID NO:229) Ser Phe Glu Ser Leu Gly Gly Leu; (SEQID NO:230) Ile Thr Lys Gly Ser Ser Phe Pro; (SEQ ID NO:231) Leu Asp TrpAla Arg Lys Leu Arg; (SEQ ID NO:232) Thr Ala Trp Asn Leu Leu Gly Tyr;(SEQ ID NO:233) Phe Gly Gln Gly Ile Lys His Val; (SEQ ID NO:234) Asp ValVal Trp Gln Arg Leu Leu; (SEQ ID NO:235) Tyr Val Asp Arg Phe Ile GlyTrp; (SEQ ID NO:236) Lys Met Ala Arg Pro Glu Gly Asn; (SEQ ID NO:237)Leu Gly Arg Trp Gly His Glu Ser; (SEQ ID NO:238) Ser Ile Trp Ser Leu LeuVal Leu; (SEQ ID NO:239) Val Trp Leu Asp Leu Leu Leu Ser; (SEQ IDNO:240) Tyr Leu Asp Thr Ser Leu Phe Gly; (SEQ ID NO:241) Thr Trp Trp ProSer Ile Thr Trp; (SEQ ID NO:242) Tyr Gly Leu Trp Trp Phe Pro Trp; (SEQID NO:243) Phe Ser Pro Ala Asp Thr Arg Tyr; (SEQ ID NO:244) Cys Asn ArgLeu Gln Ile Asp Cys; (SEQ ID NO:245) Ser Leu Val Ala Ala Arg Asn Leu;(SEQ ID NO:246) Phe Thr Ile His Asn Val Ala Val; (SEQ ID NO:247) Met GlyPro Leu Gly Pro Leu Leu; (SEQ ID NO:248) Arg Gln Leu Ser Glu Leu PheVal; (SEQ ID NO:249) Arg Val Val Cys Gln Ala Leu Leu; (SEQ ID NO:250)Trp Pro His Leu Trp Trp Leu Asp; (SEQ ID NO:251) Trp Met Asp Trp Val TrpHis Thr; (SEQ ID NO:252) Trp Trp Gly Tyr Leu Ile Cys Gln; (SEQ IDNO:253) Phe Arg Gly Leu Ser Glu Gly Pro; (SEQ ID NO:254) Ser Trp Phe AspTrp Leu Val Ala; (SEQ ID NO:255) Val Val Met Trp Tyr Ser Val Asp; (SEQID NO:256) Trp Gly Trp Ser Leu Ala Thr; (SEQ ID NO:257) Leu Gly Trp PheAsp Arg Phe Phe; (SEQ ID NO:258) Ala Trp Trp Trp Pro Thr Tyr Val; (SEQID NO:259) Gly Phe Leu Ser Ser Trp Phe Leu; (SEQ ID NO:260) Gly Val IleAsn Cys Ala Gly Thr; (SEQ ID NO:261) Val Cys Ala Arg Ala Ala His Leu;(SEQ ID NO:262) Gly Asn Ser Tyr Gly Asp Gly Gly; (SEQ ID NO:263) Gly PheLeu Ser Ser Trp Phe Leu; (SEQ ID NO:264) Phe Asp Gln Pro Gly Arg PheLeu; (SEQ ID NO:265) Arg Ser His Ala Thr Gly Val Val; (SEQ ID NO:266)Gly Tyr Trp Ala Met Met Ser Trp; (SEQ ID NO:267) Cys His Ser Met Trp AspGly Leu; (SEQ ID NO:268) Phe Ile Trp Arg Gly Trp Pro His; (SEQ IDNO:269) Leu Ser Phe Leu Gly Gly Arg Leu; (SEQ ID NO:270) Phe Ser Gly ValArg Gln Pro Asn; (SEQ ID NO:271) Trp Gly Trp Met Pro Phe Tyr Tyr; (SEQID NO:272) Phe Thr Arg Pro Ala Val Val Asp; (SEQ ID NO:273) Asp Leu TrpThr Trp Leu Gly Leu; (SEQID NO:274) Cys Asp Thr Ala Ala Val Ala Asp;(SEQ ID NO:275) Trp Trp Val Lys His His Met Leu; (SEQ ID NO:276) Ile AlaPhe Leu Arg Asp Asn Arg; (SEQ ID NO:277) Leu Ala Arg Pro Asp His TyrSer; (SEQ ID NO:278) Met Glu Ser Lys Arg Trp Thr Val; (SEQ ID NO:279)Met Ile Leu Lys Gly Tyr Ser Arg; (SEQ ID NO:280) Ala Pro Ser Asp Tyr AspGlu Ser; (SEQ ID NO:281) His Trp Leu Arg Ser Lys Arg Thr; (SEQ IDNO:282) Gly Ala Arg Val Trp Asn Tyr Gln; (SEQ ID NO:283) Leu Ser Asn TrpAsn Met Arg Leu; (SEQ ID NO:284) Cys Gly Ala Ala Gln Gln Gly Met; (SEQID NO:285) Gly Ser Ser Met Val Val Gln Arg;. (SEQ ID NO:286)

[0052] Using this technique, Blond-Elguindi have concluded that the heatshock protein BiP recognizes polypeptides that contain a heptamericregion having the sequence

[0053] Hy(Trp/X)HyXHyXHy

[0054] where Hy represents a hydrophobic amino acid residue (SEQ IDNO:29), particularly tryptophan, leucine or phenylalanine (SEQ IDNO:30), and X is any amino acid. High affinity heat-shockprotein-binding sequences incorporating this motif include: His Trp AspPhe Ala Trp Pro Trp (SEQ ID NO:1); and Phe Trp Gly Leu Trp Pro Trp Glu(SEQ ID NO:4).

[0055] Other heat shock protein binding motifs have also beenidentified. For example, Auger et al., Nature Medicine 2:306-310 (1996)have identified two pentapeptide binding motifs Gln Lys Arg Ala Ala and(SEQ ID NO:5) Arg Arg Arg Ala Ala (SEQ ID NO:6)

[0056] in HLA-DR types associated with rheumatoid arthritis which bindto heat shock proteins. Heat shock protein binding motifs have also beenidentified as consisting of seven to fifteen residue long peptides whichare enriched in hydrophobic amino acids. Lys Arg Gln Ile Tyr Thr Asp LeuGlu Met Asn Arg Leu Gly Lys; (SEQ ID NO:287) Leu Ser Ser Leu Phe Arg ProLys Arg Arg Pro Ile Tyr Lys Ser; (SEQ ID NO:288) Lys Leu Ile Gly Val LeuSer Ser Leu Phe Arg Pro Lys; (SEQ ID NO:289) Arg Arg Pro Ile Tyr Lys SerAsp Val Gly Met Ala His Phe Arg; (SEQ ID NO:290) Cys Lys Ile Gln Ser ThrPro Val Lys Gln Ser; (SEQ ID NO:291) Glu Gly Met Ile Asp Gly Trp Tyr GlyPhe Arg His Gln Asn Cys; (SEQ ID NO:292) Val Gly Ile Asp Leu Gly Thr ThrTyr Ser Cys; (SEQ ID NO:293) Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys;(SEQ ID NO:294) (Flynn et al., Science 245: 385-390 (1989)), Gly Lys TrpVal Tyr Ile; (SEQ ID NO:295) Ala Lys Arg Glu Thr Lys; (SEQ ID NO:296)Lys Trp Val His Leu Phe; (SEQ ID NO:297) Arg Leu Val Leu Val Leu; (SEQID NO:298) Trp Lys Trp Gly Ile Tyr; (SEQ ID NO:299) Ser Ser His Ala SerAla; (SEQ ID NO:300) Trp Gly Pro Trp Ser Phe; (SEQ ID NO:301) Ala IlePro Gly Lys Val; (SEQ ID NO:302) Arg Val His Asp Pro Ala; (SEQ IDNO:303) Arg Ser Val Ser Ser Phe; (SEQ ID NO:304) Leu Gly Thr Arg LysGly; (SEQ ID NO:305) Lys Asp Pro Leu Phe Asn; (SEQ ID NO:306) Leu SerGln His Thr Asn; (SEQ ID NO:307) Asn Arg Leu Leu Leu Thr; (SEQ IDNO:308) Tyr Pro Leu Trp Val Ile; (SEQ ID NO:309) Leu Leu Ile Ile AspArg; (SEQ ID NO:310) Arg Val Ile Ser Leu Gln; (SEQ ID NO:311) Glu ValSer Arg Glu Asp; (SEQ ID NO:312) Ser Ile Leu Arg Ser Thr; (SEQ IDNO:313) Pro Gly Leu Val Trp Leu; (SEQ ID NO:314) Val Lys Lys Leu TyrIle; (SEQ ID NO:315) Asn Asn Arg Leu Leu Asp; (SEQ ID NO:316) Ser LysGly Arg Trp Gly; (SEQ ID NO:317) Ile Arg Pro Ser Gly Ile; (SEQ IDNO:318) Ala Ser Leu Cys Pro Thr; (SEQ ID NO:319) Asp Val Pro Gly LeuArg; (SEQ ID NO:320) Arg His Arg Glu Val Gln; (SEQ ID NO:321) Leu AlaArg Lys Arg Ser; (SEQ ID NO:322) Ser Val Leu Asp His Val; (SEQ IDNO:323) Asn Leu Leu Arg Arg Ala; (SEQ ID NO:324) Ser Gly Ile Ser AlaTrp; (SEQ ID NO:325) Phe Tyr Pro Trp Val Arg; (SEQ ID NO:326) Lys LeuPhe Leu Pro Leu; (SEQ ID NO:327) Thr Pro Thr Leu Ser Asp; (SEQ IDNO:328) Thr His Ser Leu Ile Leu; (SEQ ID NO:329) Leu Leu Leu Leu SerArg; (SEQ ID NO:330) Leu Leu Arg Val Arg Ser; (SEQ ID NO:331) Glu ArgArg Ser Arg Gly; (SEQ ID NO:332) Arg Met Leu Gln Leu Ala; (SEQ IDNO:333) Arg Gly Trp Ala Asn Ser; (SEQ ID NO:334) Arg Pro Phe Tyr SerTyr; (SEQ ID NO:335) Ser Ser Ser Trp Asn Ala; (SEQ ID NO:336) Leu GlyHis Leu Glu Glu; (SEQ ID NO:337) Ser Ala Val Thr Asn Thr; (SEQ IDNO:338) Leu Arg Arg Ala Ser Leu; (SEQ ID NO:339) Leu Arg Arg Trp SerLeu; (SEQ ID NO:340) Lys Trp Val His Leu Phe; (SEQ ID NO:341) Asn ArgLeu Leu Leu Thr; (SEQ ID NO:342) Ala Arg Leu Leu Leu Thr; (SEQ IDNO:343) Asn Ala Leu Leu Leu Thr; (SEQ ID NO:344) Asn Arg Leu Ala LeuThr; (SEQ ID NO:345) Asn Leu Leu Arg Leu Thr; (SEQ ID NO:346) Asn ArgLeu Trp Leu Thr; (SEQ ID NO:347) Asn Arg Leu Leu Leu Ala; and (SEQ IDNO:348) Met Gln Glu Arg Ile Thr Leu Lys Asp Tyr Ala Met (SEQ ID NO:349)(Gragerov et al., J. Molec. Biol. 235:848-854 (1994)).

[0057] The hybrid antigen of the invention incorporates one immunogenicdomain and one heat shock protein-binding domain, optionally separatedby a short peptide linker. The hybrid peptide of the invention may besynthesized using chemical peptide synthesis methods or it can besynthesized by expression of a nucleic acid construct containing linkedsequences encoding the antigenic and heat shock protein binding domains.One suitable technique utilizes initial separate PCR amplificationreactions to produce separate DNA segments encoding the two domains,each with a linker segment attached to one end, followed by fusion ofthe two amplified products in a further PCR step. This technique isreferred to as linker tailing. Suitable restriction sites may also beengineered into regions of interest, after which restriction digestionand ligation is used to produce the desired hybrid peptide-encodingsequence.

[0058] Methods of Administration

[0059] The heat shock protein/target antigen combinations of theinvention may be administered to a subject using either a protein-basedor nucleic acid vaccine, so as to produce, in the subject, an amount ofheat shock protein/target antigen complex which is effective in inducinga therapeutic immune response in the subject.

[0060] The subject may be a human or nonhuman subject.

[0061] The term “therapeutic immune response,” as used herein, refers toan increase in humoral and/or cellular immunity, as measured by standardtechniques, which is directed toward the target antigen. Preferably, butnot by way of limitation, the induced level of humoral immunity directedtoward target antigen is at least four-fold, and preferably at least16-fold greater than the levels of the humoral immunity directed towardtarget antigen prior to the administration of the compositions of thisinvention to the subject. The immune response may also be measuredqualitatively, by means of a suitable in vitro or in vivo assay, whereinan arrest in progression or a remission of neoplastic or infectiousdisease in the subject is considered to indicate the induction of atherapeutic immune response.

[0062] Specific amounts of heat shock protein/target antigenadministered may depend on numerous factors including the immunogenicityof the particular vaccine composition, the immunocompetence of thesubject, the size of the subject and the route of administration.Determining a suitable amount of any given composition foradministration is a matter of routine screening.

[0063] In specific nonlimiting embodiments of the invention, it may bedesirable to include more than one species of heat shock protein, and/ormore than one target antigen, in order to optimize the immune response.Such an approach may be particularly advantageous in the treatment ofcancer or in the treatment of infections characterized by the rapiddevelopment of mutations that result in evasion of the immune response.

[0064] In other specific nonlimiting embodiments of the invention, inorder to promote binding among members of each heat shock protein/targetantigen pair, the ratio of heat shock protein to target antigen maypreferably be 1:2 to 1:200. Higher relative levels of antigen aresuitable to enhance binding to the heat shock protein.

[0065] According to still further specific but nonlimiting embodimentsof the invention, the target antigen is not chemically cross-linked tothe heat shock protein.

[0066] Compositions comprising target antigen/heat shock protein as setforth above are referred to herein as “vaccines.” The term vaccine isused to indicate that the compositions of the invention may be used toinduce a therapeutic immune response.

[0067] A vaccine composition comprising one or more heat shock proteinsand one or more target antigens in accordance with the invention may beadministered cutaneously, subcutaneously, intravenously,intramuscularly, parenterally, intrapulmonarily, intravaginally,intrarectally, nasally or topically. The vaccine composition may bedelivered by injection, particle bombardment, orally or by aerosol.

[0068] Incubation of heat shock proteins in solution with the targetantigen is sufficient to achieve loading of the antigen onto the heatshock protein in most cases. It may be desirable in some cases, however,to add agents which can assist in the loading of the antigen.

[0069] Incubation with heating of the heat shock protein with the targetantigen will in general lead to loading of the antigen onto the heatshock protein. In some cases, however, it may be desirable to addadditional agents to assist in the loading. For example, hsp40 canfacilitate loading of peptides onto hsp70. Minami et al., J. Biol. Chem.271:19617-19624 (1996). Denaturants such as guanidinium HCl or urea canbe employed to partially and reversibly destabilize the heat shockprotein to make the peptide binding pocket more accessible to theantigen.

[0070] Vaccine compositions in accordance with the invention may furtherinclude various additional materials, such as a pharmaceuticallyacceptable carrier. Suitable carriers include any of the standardpharmaceutically accepted carriers, such as phosphate buffered salinesolution, water, emulsions such as an oil/water emulsion or atriglyceride emulsion, various types of wetting agents, tablets, coatedtablets and capsules. An example of an acceptable triglyceride emulsionuseful in intravenous and intraperitoneal administration of thecompounds is the triglyceride emulsion commercially known asIntralipid®. Typically such carriers contain excipients such as starch,milk, sugar, certain types of clay, gelatin, stearic acid, talc,vegetable fats or oils, gums, glycols, or other known excipients. Suchcarriers may also include flavor and color additives or otheringredients.

[0071] The vaccine composition of the invention may also includesuitable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions may be in the form of liquid orlyophilized or otherwise dried formulations and may include diluents ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength, additives such as albumin or gelatin to preventabsorption to surfaces, detergents (e,g., Tween 20, Tween 80, PluronicF68, bile acid salts), solubilizing agents (e.g. glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulkingsubstances or tonicity modifiers (e.g., lactose, mannitol), covalentattachment of polymers such as polyethylene glycol to the protein,complexing with metal ions, or incorporation of the material into oronto particulate preparations of polymeric compounds such as polylacticacid, polyglycolic acid, hydrogels, etc. or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance. The choice of compositions will depend on thephysical and chemical properties of the vaccine. For example, a productderived from a membrane-bound form of a protein may require aformulation containing detergent. Controlled or sustained releasecompositions include formulation in lipophilic depots (e.g. fatty acids,waxes, oils). Also comprehended by the invention are particulatecompositions coated with polymers (e.g. poloxamers or poloxamines) andcoupled to antibodies directed against tissue-specific receptors,ligands or antigens or coupled to ligands of tissue-specific receptors.Other embodiments of the compositions of the invention incorporateparticulate forms protective coatings, protease inhibitors or permeationenhancers for various routes of administration, including intramuscular,parenteral, pulmonary, nasal and oral.

[0072] As an alternative to direct administration of the heat shockprotein and target antigen, one or more polynucleotide constructs may beadministered which encode heat shock protein and target antigen inexpressible form. The expressible polynucleotide constructs areintroduced into cells in the subject using ex vivo or in vivo methods.Suitable methods include injection directly into tissue and tumors,transfecting using liposomes (Fraley et al., Nature 370:111-117 (1980)),receptor-mediated endocytosis (Zatloukal et al., Ann. NY Acad. Sci.660:136-153 (1992)), particle bombardment-mediated gene transfer(Eisenbraun et al., DNA & Cell Biol. 12:792-797 (1993)) and transfectionusing peptide presenting bacteriophage (Barry et al, Nature Medicine2:299-305 (1996)). The polynucleotide vaccine may also be introducedinto suitable cells in vitro which are then introduced into the subject.

[0073] To construct an expressible polynucleotide, a region encoding theheat shock protein and/or target antigen is prepared as discussed aboveand inserted into a mammalian expression vector operatively linked to asuitable promoter such as the SV40 promoter, the cytomegalovirus (CMV)promoter or the Rous sarcoma virus (RSV) promoter. The resultingconstruct may then be used as a vaccine for genetic immunization. Thenucleic acid polymer(s) could also be cloned into a viral vector.Suitable vectors include but are not limited to retroviral vectors,adenovirus vectors, vaccinia virus vectors, pox virus vectors andadenovirus-associated vectors. Specific vectors which are suitable foruse in the present invention are pCDNA3 (InVitrogen), plasmid AH5 (whichcontains the SV40 origin and the adenovirus major late promoter),pRC/CMV (InVitrogen), pCMU II (Paabo et al., EMBO J. 5:1921-1927(1986)), pZip-Neo SV (Cepko et al., Cell 37:1053-1062 (1984)) and pSRa(DNAX, Palo Alto, Calif.).

EXAMPLE 1 Preparation of Hybrid Peptides

[0074] Hybrid peptides containing a BiP-binding domain(His-Trp-Asp-Phe-Ala-Trp-Pro-Trp; SEQ ID NO:1) and an OVA antigenicdomain (Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu; SEQ ID NO:7) separated by atripeptide linker (gly-ser-gly) were synthesized. Peptides were producedin both orientations, OVA-BiP-binding domain and BiP-binding domain-OVAas follows:Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-Gly-Ser-Gly-His-Trp-Asp-Phe-Ala-Trp-Pro-Trp(SEQ ID NO:8) andHis-Trp-Asp-Phe-Ala-Trp-Pro-Trp-Gly-Ser-Gly-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu(SEQ ID NO:9).

EXAMPLE 2

[0075] Purified mouse cytosolic hsp70 was prepared from E. coli DH5αcells transformed with pMS236 encoding mouse cytosolic hsp70. The cellswere grown to an optical density (600 nm) of 0.6 at 37° C., andexpression was induced by the addition of IPTG to a final concentrationof 1 mM. Cells were harvested by centrifugation 2 to 5 hours postinduction and the pellets were resuspended to 20 ml with Buffer A (20 mMHepes pH 7.0, 25 mM KCl, 1 mM DTT, 10 mM (NH₄)₂SO₄, 1 mM PMSF). Thecells were lysed by passing three times through a French press. Thelysate was cleared by a low speed spin, followed by centrifugation at100,000×G for 30 minutes. The cleared lysate was applied to a PharmaciaXK26 column packed with 100 ml DEAE Sephacel and equilibrated withBuffer A at a flow rate of 0.6 cm/min. The column was washed to stablebaseline with Buffer A and eluted with Buffer A adjusted to 175 mM KCl.The eluate was applied to a 25 ml ATP-agarose column (Sigma A2767),washed to baseline with Buffer A, and eluted with Buffer A containing 1mM MgATP preadjusted to pH 7.0. EDTA was added to the eluate to a finalconcentration of 2 mM. The eluate which contained essentially pure hsp70was precipitated by addition of (NH₄)₂SO₄ to 80% saturation. Theprecipitate was resuspended in Buffer A containing 1 mM MgCl₂ anddialyzed against the same buffer with multiple changes. The purifiedhsp70 was frozen in small aliquots at −70° C.

EXAMPLE 3

[0076] The purified hsp70 was combined with the synthesized peptides andused for immunization. To form the hsp70/peptide mixtures, approximately15 μg (21.5 μM) hsp70 was combined with 5 μg of Ova-peptide (0.5 mM; SEQID NO:7) or 10 μg (0.5 mM) hybrid peptide (SEQ ID NOS:8 and 9) weremixed on ice to a final volume of 10 μl in Buffer B (finalconcentration: 20 mM Hepes pH 7.0, 150 mM KCl, 10 mM (NH₄)₂SO₄, 2 mMMgCl₂ and 2 mM MgADP, pH 7.0). The mixtures were incubated for 30minutes at 37° C. and then used for in vivo immunizations.

[0077] C57BL/6 mice were immunized intradermally once a week for a totalof two weeks with 10 μl of one of the mixtures described above or with amixture of TITERMAX® (Vaxcell, Norcross, Ga.) and Ova-peptide (5 μg).One week after the second immunization, spleen cells were removed andmononuclear cells (6-8×10⁷) were cultured with 3×10⁶ γ-irradiated (3000rad) stimulator cells. The stimulator cells were obtained from naivemice that had been sensitized in vitro with Ova-peptide (10 mg/ml) for30 minutes at room temperature, washed and irradiated at 3000 rads.

[0078] Cytotoxicity of spleen cells from vaccinated mice were assayed onOva-peptide pulsed EL4 cells in an 18-hour chromium release assay. CTLwere generated by culturing in vivo immunized spleen cells for 5 days ata concentration of 10⁶ cells/ml in RPMI medium, 10% FCS, penicillin,streptomycin and 2 mM L-glutamine, together with 3×10⁶ γ-irradiated(3,000 rad) stimulator cells/ml. Target cells were prepared by culturingcells for 1 hour in the presence of 250 μCi of ⁵¹Cr sodium chromate(DuPont, Boston, Mass.) in Tris-phosphate buffer, pH 7.4 at 37° C. for60 minutes. After washing, 10⁴ ⁵¹Cr-labeled target cells were mixed witheffector lymphocytes to yield several different effector/target (E/T)ratios and were incubated for 18 hours. Supernatants were harvested andthe radioactivity was measured in a gamma counter. Percent specificlysis was calculated as: 100×[(cpm release by CTL−cpm spontaneousrelease)/(cpm maximal release−cpm spontaneous release)]. Maximalresponse was determined by addition of 1% Triton X-100. Spontaneousrelease of all target in the absence of effector cells was less than 25%of the maximal release.

[0079] As shown in FIG. 1, the combination of Hsp70 and the hybridpeptide of either orientation (hsp70+BiP-OVA or hsp70+OVA-BiP) evoked ahigher immune response as measured by specific lysis of cells than thehsp70 or TITERMAX® adjuvant plus Ova-peptide alone.

EXAMPLE 4

[0080] The assay of Example 3 was repeated using CTL cell lines whichhad been maintained by stimulation with irradiated stimulators,syngeneic splenic feeder cells plus T cell growth factors for a periodof two weeks. As shown in FIG. 2, the combination of hsp70 and thehybrid peptide of either orientation (hsp70+BiP-OVA or hsp70+OVA-BiP)evoked a higher immune response as measured by specific lysis of cellsthan the hsp70 or TITERMAX® adjuvant plus Ova-peptide alone. Thus, theimmune response elicited by the hybrid peptides persisted throughadditional passages and can be maintained over a period of time.

EXAMPLE 5

[0081] The experiment of Example 2 was repeated for the combinations ofhsp70 plus BiP-OVA and TITERMAX® plus OVA peptide using only a singleimmunization one week before removal of the spleen cells. As shown inFIG. 3, the single immunization with either composition was effective ineliciting a cellular immune response.

EXAMPLE 6

[0082] The assay of Example 3 was repeated using mixtures of TITERMAX®with Ova-peptide or the hybrid peptides of Example 1. As shown in FIG.4, no significant difference was observed between the Ova-peptide andhybrid peptides demonstrating the specificity of the effect when hybridpeptides are used in association with the heat shock protein.

EXAMPLE 7

[0083]FIGS. 5A and 5B show the results when the procedure of Example 3was repeated immunizing the mice with hsp70 alone, Ova-peptide alone,OVA-BiP alone or BiP-OVA alone. As shown, the results in all cases werethe same when the cells were pulsed with Ova-peptide (FIG. 5A) and whenthey had not been pulsed. (FIG. 5B). This demonstrates that the responseis the result of the combination of the mixture of the antigen(Ova-peptide or hybrid peptide) and the heat shock protein and not toany of the components individually.

EXAMPLE 8

[0084]¹⁴C-labeled OVA-BiP was prepared by alkylation of OVA-BiP with¹⁴C-formaldehyde. 0.9 mg of OVA-BiP in 300 μl 10% DMSO/water was addedto 175 μl of ¹⁴C-formaldehyde (62 μCi) and immediately 50 μl of freshlymade up 200 mM NaCNBH₃ was added. The reaction was mixed and left at 25°C. for 3 hours. The labeled peptide was repurified by reverse phase HPLCon a C-4 column in a 15 minute 0-100% acetonitrile (0.1% TFA) gradient.

[0085] The ability of the OVA-BiP peptide to bind to heat shock proteinswas measured by incubating 100 μM (5 μg) ¹⁴C-labeled OVA-BiP with 50 μgof BiP (prepared as in Example 11), hsp70 (as prepared in Example 2) orgp96 (prepared as in Example 10) in a final volume of 20 μl of buffer(50 mM Mops, pH 7.2, 200 mM NaCl, 5 mM MgAcetate) at 37° C. for 30minutes. The samples were then spun down (5 minutes in a microfuge) andloaded onto a 17 cm long Sephacryl S-300 column equilibrated in bindingbuffer (50 mM Mops, pH 7.2, 200 mM NaCl, 5 mM MgAcetate) and fractionswere collected dropwise. 50 μl of each ˜225 μl fraction was counted inscintillation liquid. 10 μl of each fraction was also run on a 12%SDS-PAGE reducing gel. FIG. 6 shows the radioactivity detected in eachfraction eluted from the column, together with the center of the peak ofheat shock protein as determined by SDS-PAGE. As shown, a significantamount of radioactivity elutes with BiP and hsp70, thus providingevidence that the hybrid peptide binds to these two heat shock proteins.The result for gp96 is unclear because the peak at fraction 11 (whichmay represent an aggregation phenomenon) and the gp96 peak (fraction 14)elute close together on the column used.

EXAMPLE 9

[0086] To prepare ¹²⁵I-OVA-BiP, 250 μCi of monoiodinated Bolton-Hunterreagent was transferred into a stoppered vial and the solvent in whichit was dissolved was evaporated with a gentle stream of argon gas. Tothe dried reagent 222 μl of 4.5 mg/ml OVA-BiP in 100 mM NaBO₃, pH 8.9,10% DMSO was added. The reaction was mixed and incubated at 25° C. for45 minutes and continued at 4° C. for a further hour. The labeledpeptide was repurified by reverse phase HPLC on a C-4 column in a 20minute, 0-100% acetonitrile (0.1% TFA) gradient.

[0087] The iodinated OVA-BIP was combined with BiP in substantially thesame manner as the heat shock proteins in Example 7, except that sincethe iodinated peptide was at a very low concentration, 1 μl (approx. 32ng) of labeled peptide was mixed with 5 μg of unlabeled peptide and thiswas incubated with 50 μg of BiP in 20 μl of binding buffer. To observeATP-mediated peptide release, ATP was added to a final concentration of2 mM after the 30 minute incubation and incubated for a further 5minutes prior to spinning. These samples were run on the same column asabove, but equilibrated in binding buffer supplemented with 2 mM ATP.

[0088]FIG. 7 shows the elution profile for a mixture of the ¹²⁵I-OVA-BiPand BiP in the presence and absence of 2 mM ATP. As shown, addition ofATP causes the release of the hybrid peptide from the BiP. This isconsistent with the observation that ATP mediates release of boundproteins or polypeptides from heat shock proteins.

EXAMPLE 10

[0089] Hybrid peptides for use in a vaccine in accordance with theinvention against human papilloma virus are prepared using a peptidesynthesizer as follows: E7 (Type 11)-BiPLeu-Leu-Leu-Gly-Thr-Leu-Asn-Ile-Val-gly-ser-gly-His- (SEQ ID NO:10)Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7 (Type 11)His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Leu-Leu- (SEQ ID NO:11)Leu-Gly-Thr-Leu-Asn-Ile-Val E7 (Type 16)-BiPLeu-Leu-Met-Gly-Thr-Leu-Gly-Ile-Val-gly-ser-gly-His- (SEQ ID NO:12)Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7 (Type 16)His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Leu-Leu- (SEQ ID NO:13)Met-Gly-Thr-Leu-Gly-Ile-Val E7 (Type 18) -BiPThr-Leu-Gln-Asp-Ile-Val-Leu-His-Leu-gly-ser-gly-His- (SEQ ID NO:14)Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7 (Type 18)His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Thr-Leu- (SEQ ID NO:15)Gln-Asp-Ile-Val-Leu-His-Leu E7.1 (Type 6b)-BiPGly-Leu-His-Cys-Tyr-Glu-Gln-Leu-Val-gly-ser-gly-His- (SEQ ID NO:16)Trp-Asp-Phe-Ala-Trp-Pro-Trp BiP-E7.1 (Type 6b)His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Gly-Leu- (SEQ ID NO:17)His-Cys-Tyr-Glu-Gln-Leu-Val E7.2 (Type 6b) -BiPPro-Leu-Lys-Gln-His-Phe-Gln-Ile-Val-gly-ser-gly-His- (SEQ ID NO:18)Trp-Asp-Phe-Ala-Trp-Pro-Trp Bip-E7.2 (Type 6b)His-Trp-Asp-Phe-Ala-Trp-Pro-Trp-gly-ser-gly-Pro-Leu- (SEQ ID NO:19)Lys-Gln-His-Phe-Gln-Ile-Val

[0090] Hybrid polypeptides for use in vaccines against human papillomavirus or other types of proteins from other viruses, bacteria etc. canbe developed by searching their sequences for MHC class I restrictedpeptide epitopes containing the HLA-A2 peptide binding motif.

EXAMPLE 11 Preparation of Recombinant GP96

[0091] The DNA sequence encoding a wild-type or KDEL deleted gp96polypeptide was subcloned from pRc/CMV into the vector pET11a (Novagen).Thus upon expression, mature gp96 could be purified from cell lysates.

[0092] Vector Construction

[0093] PCR amplification of the sequence encoding gp96 (from pRc/CMV)was performed with the following primers. The 5′ primer for bothwild-type and KDEL-deleted gp96 was complementary to the DNA sequenceencoding the amino terminal end of the mature form of gp96 and an Nde Irestriction site (CATATC) the ATC of which forms the initiator codon:

[0094] 5′ AGA TAT ACA TAT GGA TGA TGA AGT CGA CGT GG 3′ (SEQ ID NO:20)

[0095] The 3′ primers were complementary to the DNA sequence of gp96encoding the carboxyl terminal end of the protein, with the nucleotidesencoding the KDEL sequence removed in the primer for the KDEL-deletedvariant. Both primers contain a BamH I restriction site (GGATCC)followed by a STOP codon as shown: Wild-type: 5′ TCG GAT CCT TAC AAT TCATCC TTC TCT GTA GAT TC 3′ (SEQ ID NO:21) KDEL-deleted: 5′ TCG GAT CCTTAC TCT GTA GAT TCC TTT TC 3′ (SEQ ID NO:22)

[0096] The PCR products were cut with Nde I and BamH I and ligated intopET11a (Novagen) which had also been cut with these enzymes. Theligation product was used to transform competent BL21 cells. Clonesobtained were screened by expression screening.

[0097] Expression and Purification

[0098] This procedure is identical for wild-type or KDEL deleted gp96.Two liters of E. coli BL21 cells transformed with pET11a containing asequence coding for either wild type or KDEL-deleted gp96 were grown in2×TY medium supplemented with 200 μg/ml ampicillin at 37° C. until theyreached an absorbance at 600 nm of 0.5-0.6 at which point they wereinduced by the addition of 1 mM IPTG. The cells were allowed to grow fora further 2-5 hours at 37° C. and then they were harvested by 10 minutescentrifugation at 7000×G. The cell pellet was resuspended in 50 mM HepespH 7.5, 50 mM KCl, 5 mM MgAcetate, 20% sucrose, 1 mM PMSF and the cellslysed by passing them through the French Press three times. The cellextract was clarified by a one hour spin at 200,000×G and thesupernatant retained.

[0099] The supernatant was diluted two-fold with cold 50 mM Hepes pH 7.5and loaded onto a Pharmacia XK26 column containing 50 ml of DE52 anionexchange resin (Whatman) which had been equilibrated in 50 mM Mops pH7.4, 10 mM NaCl, 5 mM MgAcetate. The bound protein was eluted in a0-1000 mM NaCl gradient. Fractions containing gp96 were identified bySDS-PAGE and pooled.

[0100] The pooled gp96-containing fractions were diluted two-fold withcold 50 mM Mops pH 7.4 and loaded onto a Pharmacia XK16 columncontaining 15 ml of hydroxylapatite resin (BioRad) which had been washedwith 0.5 M K₂HPO₄ pH 7.2, 50 mM KCl and equilibrated in 10 mM K₂HPO₄ pH7.2, 50 mM KCl. The bound protein was eluted in a 10-500 mM K₂HPO₄ pH7.2 gradient with the KCl concentration held constant at 50 mM.Fractions containing gp96 were identified by SDS-PAGE and pooled.

[0101] The pooled gp96-containing fractions were finally loaded onto aPharmacia XK26 column containing 25 ml of phenyl Sepharose (Pharmacia)which had been equilibrated in 50 mM Mops pH 7.2, 500 mM NaCl and elutedin a 500-0 mM NaCl gradient. The fractions containing essentially puregp96 were pooled, concentrated by filtration and made up to 10%glycerol. The purified gp96 was stored frozen at −80° C.

EXAMPLE 12 Construction of BiP Expression Vector and Purification ofRecombinant BiP

[0102] The DNA sequence encoding the wild-type or KDEL-deleted BiPpolypeptide was subcloned from pCDNA3 into the vector pET22 (Novagen),thereby placing it behind and in frame with a DNA sequence that codesfor a signal sequence which targets the expressed BiP to the periplasmicspace of the bacterial expression host, E. coli. Upon transport into theperiplasm, the signal sequence is removed and thus mature wild-type orKDEL-deleted BiP can be harvested from the periplasm without anycontamination by cytosolic hsp70s.

[0103] Vector Construction:

[0104] PCR amplification of the sequence encoding BiP (from pCDNA3) wasperformed with the following primers. The 5′ primer for both wild-typeand KDEL-deleted BiP was complementary to the DNA sequence of BiPencoding the amino terminal end of the mature form of BiP with an Msc Irestriction site (TGGCCA) immediately upstream from the initiator ATGcodon.

[0105] 5′ AGA TAT GTG GCC ATG GAG GAG GAG GAC AAG 3′ (SEQ ID NO:23) The3′ primers were complementary to the DNA sequence of BiP encoding thecarboxyl terminal end of the protein, with the nucleotides encoding theKDEL sequence removed in the primer for the KDEL-deleted variant. Bothprimers contain a BamH I restriction site (GGATCC) followed by stopcodon as shown: Wild-type: (SEQ ID NO:24) 5′ TCG GAT CCC TAC AAC TCA TCTTTT TCT G 3′ KDEL-deleted: (SEQ ID NO:25) 5′ TCG GAT CCC TAT TCT GAT GTATCC TCT TCA CC 3′

[0106] The PCR products were cut with Msc I and BamH I and ligated intopET22 (Novagen) which had also been cut with these enzymes. The ligationproduct was used to transform competent BL21 cells. Clones obtained werescreened by expression screening.

[0107] Expression and Purification

[0108] The procedure is identical for wild-type or KDEL deleted BiP. Twoliters of BL21 cells transformed with pET22 containing a sequence codingfor either wild-type or KDEL deleted BiP were grown in 2×TY mediumsupplemented with 200 μg/ml ampicillin at 37° C. until they reached anabsorbance at 600 nm of 0.5-0.6 at which point they were induced by theaddition of 1 mM IPTG. The cells were allowed to grow for a further 2-5hours at 37° C. and then they were harvested by 10 minutescentrifugation at 7000×G. The cell pellet was gently resuspended in 400ml (or 80 ml/gm cells) of 30 mM Tris pH 8.0, 20% sucrose, 1 mM PMSF.Following resuspension of the cells EDTA was added to 1 mM and thesuspension incubated at room temperature for 5 minutes. The cells werethen spun down for 15 minutes at 7000×G and resuspended in 400 ml of icecold 5 mM MgSO₄, 1 mM PMSF and incubated at 4° C. for 10 minutes. Thecells were then spun down once again and the supernatant kept since thisnow constitutes the periplasmic extract.

[0109] The periplasmic extract was loaded onto a Pharmacia XK26 columncontaining 25 ml of DE52 anion exchange resin (Whatman) which had beenequilibrated in 50 mM Mops pH 7.4, 10 mM NaCl. The bound protein waseluted in a 10-500 mM NaCl gradient. Fractions containing eluted BiPwere identified by SDS-PAGE and pooled. The pooled BiP was subsequentlyrun onto a Pharmacia XK26 column containing 10 ml of ATP agarose whichhad been equilibrated in 50 mM Mops pH 7.4, 100 mM NaCl, 5 mM MgAcetate,10 mM KCl. After loading the pooled BiP containing fractions the columnwas washed until the baseline of absorption at 280 nm reached zero.Finally the bound BiP was eluted with the same buffer supplemented with1 mM ATP. The eluate was concentrated by filtration, made up to 10%glycerol and stored frozen at −80° C.

EXAMPLE 13 Preparation of Recombinant Mouse HSP40

[0110] Plasmid Constructions

[0111] The DNA fragment used to introduce an Nde I site at theinitiation methionine of hsp40 was constructed via polymerase chainreaction (PCR) using an Nde-primer

[0112] 5′-CCGCAGGAGGGGCATATGGGTAAAGAC-3′ (SEQ ID NO:26) and anNco-primer

[0113] 5′-GAGGGTCTCCATGGAATGTGTAGCTG-3′ (SEQ ID NO:27).

[0114] The latter included an Nco I site corresponding to nucleotide 322of the human hsp40 cDNA clone, pBSII-hsp40, Ohtsuka, K., Biochem.Biophys. Res. Commun. 197: 235-240 (1991), which was used as thetemplate. The Hsp40-coding region of pBSII-hsp40 was digested with BamHI and Sac I and inserted into the complementary sites in a modified formof the plasmid pET-3a (Novagen, Inc.). The PCR-amplified DNA wasdigested with Nde I and Nco I, and replaced the Nde I-Nco I region ofthe above plasmid to create the plasmid pET/hsp40, expressing hsp40.

[0115] Protein Purification.

[0116] To purify recombinant human hsp40, the plasmid pET/hsp40 wastransformed into BL21(DE3) cells grown at 37 C. After a 2 hourincubation with 0.4 mM isopropyl thio-b-D-galactoside (IPTG), cells werelysed in a French Pressure Cell (SLM Instruments, Inc.) in buffer A (20mM Tris-HCl, pH 7.5, 20 mM NaCl, 1 mM EDTA) containing 1 mM PMSF. Thecleared lysate was mixed with DEAE-Sephacel (Pharmacia) on ice for 1 h.The unbound material was collected and the resin was washed with bufferA. The flow-through and first wash were combined and loaded onto ahydroxyapatite HTP column (Bio-Rad) equilibrated with 100 mM potassiumphosphate, pH 7.6. The column was washed with the same buffer and Hsp40was eluted with a linear gradient of 100-300 mM potassium phosphate, pH7.6. Peak fractions were rechromatographed on an HTP column afterpassing them through a DEAE-Sephacel column.

EXAMPLE 14

[0117] Vaccine compositions were prepared by combining recombinant mousehsp70 (prepared as in example 2), recombinant human hsp40 (prepared asin example 13) and Ova-peptide

[0118] Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SEQ ID NO:7)

[0119] in a final volume of 10 μl of buffer (20 mM Hepes pH 7.0, 150 mMKCl, 10 mM (NH₄)₂SO₄, 2 mM MgCl₂ and 2 mM MgADP) as follows: Samplehsp70 Hsp40 ova OVA alone nil nil 5 μg Hsp70/40 15 μg 8 μg NilHsp70/40 + OVA 15 μg 8 μg 5 μg Hsp70 + OVA 15 μg 5 μg

[0120] The mixtures were incubated for 30 minutes at 37° C. prior to usefor immunizations.

[0121] C57BL/6 mice were immunized intradermally once a week for a totalof two weeks with 10 μl of one of the mixtures' described above or witha mixture of TITERMAX® (Vaxcell, Norcross, Ga.) and Ova-peptide (5 μg).One week after the second immunization, spleen cells were removed andmononuclear cells (6-8×10⁷) were cultured with 3×10⁶ γ-irradiated (3000rad) stimulator cells. The stimulator cells were obtained from naivemice that had been sensitized in vitro with Ova-peptide (10 mg/ml) for30 minutes at room temperature, washed and irradiated at 3000 rads.

[0122] Cytotoxicity of spleen cells from vaccinated mice was assayed onOva-peptide pulsed EL4 cells in an 18-hour chromium release assay. CTLwere generated by culturing in vivo immunized spleen cells for 5 days ata concentration of 10⁶ cells/ml in RPMI medium, 10% FCS, penicillin,streptomycin and 2 mM L-glutamine, together with 3×10⁶ γ-irradiated(3,000 rad) stimulator cells/ml. Target cells were prepared by culturingcells for 1 hour in the presence of 250 μCi of ⁵¹Cr sodium chromate(DuPont, Boston, Mass.) in Tris-phosphate buffer, pH 7.4 at 37° C. for60 minutes. After washing, 10⁴ ⁵¹Cr-labeled target cells were mixed witheffector lymphocytes to yield several different effector/target (E/T)ratio and were incubated for 18 hours. Supernatants were harvested andthe radioactivity was measured in a gamma counter. Percent specificlysis was calculated as: 100×[cpm release by CTL−cpm spontaneousrelease)/(cpm maximal release−cpm spontaneous release)]. Maximalresponse was determined by addition of 1% Triton X-100. Spontaneousrelease of all target in the absence of effector cells was less than 25%of the maximal release.

[0123] The results of this study are shown in FIG. 8. As shown,combinations of antigen with hsp70 or a mixture of hsp70 and hsp40 areeffective to produce a CTL response to the antigen, while theadministration of the antigen alone or a combination of heat shockproteins is not.

EXAMPLE 15

[0124] The experiment of Example 14 was repeated using EG7 lymphomacells, Moore et al., Cell 54:777-785 (1988), in place of the EL4 cells.The results are shown in FIG. 9 and are comparable to those observedusing EL4 cells.

[0125] Various publications are cited herein, the contents of which arehereby incorporated by reference in their entireties.

1 349 1 8 PRT Artificial Sequence synthetic peptide 1 His Trp Asp PheAla Trp Pro Trp 5 2 9 PRT Artificial Sequence HLA-A2 binding motif 2 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 5 3 9 PRT Artificial Sequence HLA-A2binding motif 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val 5 4 8 PRT ArtificialSequence synthetic peptide 4 Phe Trp Gly Leu Trp Pro Trp Glu 5 5 5 PRTArtificial Sequence synthetic peptide 5 Gln Lys Arg Ala Ala 5 6 5 PRTArtificial Sequence synthetic peptide 6 Arg Arg Arg Ala Ala 5 7 8 PRTArtificial Sequence synthetic peptide 7 Ser Ile Ile Asn Phe Glu Lys Leu5 8 19 PRT Artificial Sequence synthetic peptide 8 Ser Ile Ile Asn PheGlu Lys Leu Gly Ser Gly His Trp Asp Phe Ala 5 10 15 Trp Pro Trp 9 19 PRTArtificial Sequence synthetic peptide 9 His Trp Asp Phe Ala Trp Pro TrpGly Ser Gly Ser Ile Ile Asn Phe 5 10 15 Glu Lys Leu 10 20 PRT ArtificialSequence synthetic peptide 10 Leu Leu Leu Gly Thr Leu Asn Ile Val GlySer Gly His Trp Asp Phe 5 10 15 Ala Trp Pro Trp 11 20 PRT ArtificialSequence synthetic peptide 11 His Trp Asp Phe Ala Trp Pro Trp Gly SerGly Leu Leu Leu Gly Thr 5 10 15 Leu Asn Ile Val 12 20 PRT ArtificialSequence synthetic peptide 12 Leu Leu Met Gly Thr Leu Gly Ile Val GlySer Gly His Trp Asp Phe 5 10 15 Ala Trp Pro Trp 13 20 PRT ArtificialSequence synthetic peptide 13 His Trp Asp Phe Ala Trp Pro Trp Gly SerGly Leu Leu Met Gly Thr 5 10 15 Leu Gly Ile Val 14 20 PRT ArtificialSequence synthetic peptide 14 Thr Leu Gln Asp Ile Val Leu His Leu GlySer Gly His Trp Asp Phe 5 10 15 Ala Trp Pro Trp 15 20 PRT ArtificialSequence synthetic peptide 15 His Trp Asp Phe Ala Trp Pro Trp Gly SerGly Thr Leu Gln Asp Ile 5 10 15 Val Leu His Leu 16 20 PRT ArtificialSequence synthetic peptide 16 Gly Leu His Cys Tyr Glu Gln Leu Val GlySer Gly His Trp Asp Phe 5 10 15 Ala Trp Pro Trp 17 20 PRT ArtificialSequence synthetic peptide 17 His Trp Asp Phe Ala Trp Pro Trp Gly SerGly Gly Leu His Cys Tyr 5 10 15 Glu Gln Leu Val 18 20 PRT ArtificialSequence synthetic peptide 18 Pro Leu Lys Gln His Phe Gln Ile Val GlySer Gly His Trp Asp Phe 5 10 15 Ala Trp Pro Trp 19 20 PRT ArtificialSequence synthetic peptide 19 His Trp Asp Phe Ala Trp Pro Trp Gly SerGly Pro Leu Lys Gln His 5 10 15 Phe Gln Ile Val 20 32 DNA ArtificialSequence amplification primer for gp96 20 agatatacat atggatgatgaagtcgacgt gg 32 21 35 DNA Artificial Sequence amplification primer forgp96 21 tcggatcctt acaattcatc cttctctgta gattc 35 22 29 DNA ArtificialSequence amplification primer for gp96 22 tcggatcctt actctgtagattccttttc 29 23 30 DNA Artificial Sequence amplification primer for BiP23 agatatgtgg ccatggagga ggaggacaag 30 24 28 DNA Artificial Sequenceamplification primer for BiP 24 tcggatccct acaactcatc tttttctg 28 25 32DNA Artificial Sequence amplification primer for BiP 25 tcggatccctattctgatgt atcctcttca cc 32 26 27 DNA Artificial Sequence amplificationprimer for hsp40 26 ccgcaggagg ggcatatggg taaagac 27 27 26 DNAArtificial Sequence amplification primer for hsp40 27 gagggtctccatggaatgtg tagctg 26 28 9 PRT Artificial Sequence HLA-A2 binding motif28 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu 5 29 7 PRT Artificial Sequenceheat shock protein-binding motif 29 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 5 30 7PRT Artificial Sequence heat shock protein-binding motif 30 Xaa Xaa XaaXaa Xaa Xaa Xaa 5 31 10 PRT Artificial Sequence synthetic peptide 31 SerGly Pro Ser Asn Thr Pro Pro Glu Ile 5 10 32 11 PRT Artificial Sequencesynthetic peptide 32 Ser Gly Val Glu Asn Pro Gly Gly Tyr Cys Leu 5 10 3310 PRT Artificial Sequence synthetic peptide 33 Lys Ala Val Tyr Asn PheAla Thr Cys Gly 5 10 34 9 PRT Artificial Sequence synthetic peptide 34Arg Pro Gln Ala Ser Gly Val Tyr Met 5 35 9 PRT Artificial Sequencesynthetic peptide 35 Phe Gln Pro Gln Asn Gly Gln Phe Ile 5 36 9 PRTArtificial Sequence synthetic peptide 36 Ile Glu Gly Gly Trp Thr Gly MetIle 5 37 10 PRT Artificial Sequence synthetic peptide 37 Thr Tyr Val SerVal Ser Thr Ser Thr Leu 5 10 38 8 PRT Artificial Sequence syntheticpeptide 38 Phe Glu Ala Asn Gly Asn Leu Ile 5 39 9 PRT ArtificialSequence synthetic peptide 39 Ile Tyr Ser Thr Val Ala Ser Ser Leu 5 40 9PRT Artificial Sequence synthetic peptide 40 Thr Tyr Gln Arg Thr Arg AlaLeu Val 5 41 9 PRT Artificial Sequence synthetic peptide 41 Cys Thr GluLeu Lys Leu Ser Asp Tyr 5 42 8 PRT Artificial Sequence synthetic peptide42 Ser Asp Tyr Glu Gly Arg Leu Ile 5 43 9 PRT Artificial Sequencesynthetic peptide 43 Glu Glu Gly Ala Ile Val Gly Glu Ile 5 44 9 PRTArtificial Sequence synthetic peptide 44 Val Ser Asp Gly Gly Pro Asn LeuTyr 5 45 9 PRT Artificial Sequence synthetic peptide 45 Ala Ser Asn GluAsn Met Glu Thr Met 5 46 9 PRT Artificial Sequence synthetic peptide 46Ala Ser Asn Glu Asn Met Asp Ala Met 5 47 10 PRT Artificial Sequencesynthetic peptide 47 Lys Leu Gly Glu Phe Tyr Asn Gln Met Met 5 10 48 9PRT Artificial Sequence synthetic peptide 48 Leu Tyr Gln Asn Val Gly ThrTyr Val 5 49 10 PRT Artificial Sequence synthetic peptide 49 Thr Tyr ValSer Val Gly Thr Ser Thr Leu 5 10 50 8 PRT Artificial Sequence syntheticpeptide 50 Phe Glu Ser Thr Gly Asn Leu Ile 5 51 9 PRT ArtificialSequence synthetic peptide 51 Val Tyr Gln Ile Leu Ala Ile Tyr Ala 5 52 9PRT Artificial Sequence synthetic peptide 52 Ile Tyr Ala Thr Val Ala GlySer Leu 5 53 9 PRT Artificial Sequence synthetic peptide 53 Gly Ile LeuGly Phe Val Phe Thr Leu 5 54 10 PRT Artificial Sequence syntheticpeptide 54 Ile Leu Gly Phe Val Phe Thr Leu Thr Val 5 10 55 9 PRTArtificial Sequence synthetic peptide 55 Ile Leu Arg Gly Ser Val Ala HisLys 5 56 9 PRT Artificial Sequence synthetic peptide 56 Glu Asp Leu ArgVal Leu Ser Phe Ile 5 57 9 PRT Artificial Sequence synthetic peptide 57Glu Leu Arg Ser Arg Tyr Trp Ala Ile 5 58 9 PRT Artificial Sequencesynthetic peptide 58 Ser Arg Tyr Trp Ala Ile Arg Thr Arg 5 59 9 PRTArtificial Sequence synthetic peptide 59 Lys Thr Gly Gly Pro Ile Tyr LysArg 5 60 9 PRT Artificial Sequence synthetic peptide 60 Phe Ala Pro GlyAsn Tyr Pro Ala Leu 5 61 9 PRT Artificial Sequence synthetic peptide 61Arg Arg Tyr Pro Asp Ala Val Tyr Leu 5 62 9 PRT Artificial Sequencesynthetic peptide 62 Asp Pro Val Ile Asp Arg Leu Tyr Leu 5 63 9 PRTArtificial Sequence synthetic peptide 63 Ser Pro Gly Arg Ser Phe Ser TyrPhe 5 64 9 PRT Artificial Sequence synthetic peptide 64 Tyr Pro Ala LeuGly Leu His Glu Phe 5 65 8 PRT Artificial Sequence synthetic peptide 65Thr Tyr Lys Asp Thr Val Gln Leu 5 66 10 PRT Artificial Sequencesynthetic peptide 66 Phe Tyr Asp Gly Phe Ser Lys Val Pro Leu 5 10 67 11PRT Artificial Sequence synthetic peptide 67 Phe Ile Ala Gly Asn Ser AlaTyr Glu Tyr Val 5 10 68 9 PRT Artificial Sequence synthetic peptide 68Tyr Pro His Phe Met Pro Thr Asn Leu 5 69 9 PRT Artificial Sequencesynthetic peptide 69 Ala Pro Thr Ala Gly Ala Phe Phe Phe 5 70 11 PRTArtificial Sequence synthetic peptide 70 Ser Thr Leu Pro Glu Thr Thr ValVal Arg Arg 5 10 71 10 PRT Artificial Sequence synthetic peptide 71 PheLeu Pro Ser Asp Phe Phe Pro Ser Val 5 10 72 9 PRT Artificial Sequencesynthetic peptide 72 Trp Leu Ser Leu Leu Val Pro Phe Val 5 73 10 PRTArtificial Sequence synthetic peptide 73 Gly Leu Ser Pro Thr Val Trp LeuSer Val 5 10 74 9 PRT Artificial Sequence synthetic peptide 74 Asp LeuMet Gly Tyr Ile Pro Leu Val 5 75 10 PRT Artificial Sequence syntheticpeptide 75 Leu Met Gly Tyr Ile Pro Leu Val Gly Ala 5 10 76 8 PRTArtificial Sequence synthetic peptide 76 Ala Ser Arg Cys Trp Val Ala Met5 77 10 PRT Artificial Sequence synthetic peptide 77 Lys Leu Val Ala LeuGly Ile Asn Ala Val 5 10 78 9 PRT Artificial Sequence synthetic peptide78 Phe Leu Arg Gly Arg Ala Tyr Gly Leu 5 79 9 PRT Artificial Sequencesynthetic peptide 79 Arg Arg Ile Tyr Asp Leu Ile Glu Leu 5 80 9 PRTArtificial Sequence synthetic peptide 80 Ile Val Thr Asp Phe Ser Val IleLys 5 81 9 PRT Artificial Sequence synthetic peptide 81 Arg Arg Arg TrpArg Arg Leu Thr Val 5 82 10 PRT Artificial Sequence synthetic peptide 82Glu Glu Asn Leu Leu Asp Phe Val Arg Phe 5 10 83 9 PRT ArtificialSequence synthetic peptide 83 Cys Leu Gly Gly Leu Leu Thr Met Val 5 84 8PRT Artificial Sequence synthetic peptide 84 Ser Ser Ile Glu Phe Ala ArgLeu 5 85 11 PRT Artificial Sequence synthetic peptide 85 Leu Tyr Arg ThrPhe Ala Gly Asn Pro Arg Ala 5 10 86 9 PRT Artificial Sequence syntheticpeptide 86 Asp Tyr Ala Thr Leu Gly Val Gly Val 5 87 9 PRT ArtificialSequence synthetic peptide 87 Leu Leu Leu Gly Thr Leu Asn Ile Val 5 88 9PRT Artificial Sequence synthetic peptide 88 Leu Leu Met Gly Thr Leu GlyIle Val 5 89 9 PRT Artificial Sequence synthetic peptide 89 Thr Leu GlnAsp Ile Val Leu His Leu 5 90 9 PRT Artificial Sequence synthetic peptide90 Gly Leu His Cys Tyr Glu Gln Leu Val 5 91 9 PRT Artificial Sequencesynthetic peptide 91 Pro Leu Lys Gln His Phe Gln Ile Val 5 92 9 PRTArtificial Sequence synthetic peptide 92 Arg Leu Val Thr Leu Lys Asp IleVal 5 93 9 PRT Artificial Sequence synthetic peptide 93 Arg Ala His TyrAsn Ile Val Thr Phe 5 94 9 PRT Artificial Sequence synthetic peptide 94Leu Leu Phe Gly Tyr Pro Val Tyr Val 5 95 10 PRT Artificial Sequencesynthetic peptide 95 Ser Ala Ile Asn Asn Tyr Ala Gln Lys Leu 5 10 96 9PRT Artificial Sequence synthetic peptide 96 His Gln Ala Ile Ser Pro ArgThr Leu 5 97 12 PRT Artificial Sequence synthetic peptide 97 Gln Met ValHis Gln Ala Ile Ser Pro Arg Thr Leu 5 10 98 9 PRT Artificial Sequencesynthetic peptide 98 Cys Lys Gly Val Asn Lys Glu Tyr Leu 5 99 9 PRTArtificial Sequence synthetic peptide 99 Gln Gly Ile Asn Asn Leu Asp AsnLeu 5 100 9 PRT Artificial Sequence synthetic peptide 100 Asn Asn LeuAsp Asn Leu Arg Asp Tyr 5 101 9 PRT Artificial Sequence syntheticpeptide 101 Ser Glu Phe Leu Leu Glu Lys Arg Ile 5 102 9 PRT ArtificialSequence synthetic peptide 102 Ser Tyr Ile Gly Ser Ile Asn Asn Ile 5 10310 PRT Artificial Sequence synthetic peptide 103 Ile Leu Gly Asn Lys IleVal Arg Met Tyr 5 10 104 9 PRT Artificial Sequence synthetic peptide 104Arg Leu Arg Pro Gly Gly Lys Lys Lys 5 105 9 PRT Artificial Sequencesynthetic peptide 105 Glu Ile Lys Asp Thr Lys Glu Ala Leu 5 106 9 PRTArtificial Sequence synthetic peptide 106 Gly Glu Ile Tyr Lys Arg TrpIle Ile 5 107 9 PRT Artificial Sequence synthetic peptide 107 Glu IleTyr Lys Arg Trp Ile Ile Leu 5 108 9 PRT Artificial Sequence syntheticpeptide 108 Arg Tyr Leu Lys Asp Gln Gln Leu Leu 5 109 10 PRT ArtificialSequence synthetic peptide 109 Arg Gly Pro Gly Arg Ala Phe Val Thr Ile 510 110 9 PRT Artificial Sequence synthetic peptide 110 Ile Val Gly LeuAsn Lys Ile Val Arg 5 111 10 PRT Artificial Sequence synthetic peptide111 Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 5 10 112 11 PRT ArtificialSequence synthetic peptide 112 Arg Leu Arg Asp Leu Leu Leu Ile Val ThrArg 5 10 113 10 PRT Artificial Sequence synthetic peptide 113 Lys ArgTrp Ile Ile Leu Gly Leu Asn Lys 5 10 114 9 PRT Artificial Sequencesynthetic peptide 114 Ser Phe Asn Cys Gly Gly Glu Phe Phe 5 115 9 PRTArtificial Sequence synthetic peptide 115 Gly Arg Ala Phe Val Thr IleGly Lys 5 116 10 PRT Artificial Sequence synthetic peptide 116 Thr ProGly Pro Gly Val Arg Tyr Pro Leu 5 10 117 10 PRT Artificial Sequencesynthetic peptide 117 Gln Val Pro Leu Arg Pro Met Thr Tyr Lys 5 10 118 9PRT Artificial Sequence synthetic peptide 118 Thr Glu Met Glu Lys GluGly Lys Ile 5 119 9 PRT Artificial Sequence synthetic peptide 119 IleLeu Lys Glu Pro Val His Gly Val 5 120 9 PRT Artificial Sequencesynthetic peptide 120 Val Glu Ala Glu Ile Ala His Gln Ile 5 121 8 PRTArtificial Sequence synthetic peptide 121 Arg Gly Tyr Val Tyr Gln GlyLeu 5 122 9 PRT Artificial Sequence synthetic peptide 122 Tyr Ser GlyTyr Ile Phe Arg Asp Leu 5 123 9 PRT Artificial Sequence syntheticpeptide 123 Val Gly Pro Val Phe Pro Pro Gly Met 5 124 8 PRT ArtificialSequence synthetic peptide 124 Ile Ile Tyr Arg Phe Leu Leu Ile 5 125 9PRT Artificial Sequence synthetic peptide 125 Lys Tyr Gly Val Ser ValGln Asp Ile 5 126 9 PRT Artificial Sequence synthetic peptide 126 IleGln Val Gly Asn Thr Arg Thr Ile 5 127 9 PRT Artificial Sequencesynthetic peptide 127 Thr Pro His Pro Ala Arg Ile Gly Leu 5 128 9 PRTArtificial Sequence synthetic peptide 128 Ser Tyr Ile Pro Ser Ala GluLys Ile 5 129 8 PRT Artificial Sequence synthetic peptide 129 Lys ProLys Asp Glu Leu Asp Tyr 5 130 8 PRT Artificial Sequence syntheticpeptide 130 Lys Ser Lys Asp Glu Leu Asp Tyr 5 131 8 PRT ArtificialSequence synthetic peptide 131 Lys Pro Asn Asp Lys Ser Leu Tyr 5 132 10PRT Artificial Sequence synthetic peptide 132 Lys Tyr Leu Lys Lys IleLys Asn Ser Leu 5 10 133 9 PRT Artificial Sequence synthetic peptide 133Tyr Glu Asn Asp Ile Glu Lys Lys Ile 5 134 9 PRT Artificial Sequencesynthetic peptide 134 Asn Tyr Asp Asn Ala Gly Thr Asn Leu 5 135 9 PRTArtificial Sequence synthetic peptide 135 Asp Glu Leu Asp Tyr Glu AsnAsp Ile 5 136 9 PRT Artificial Sequence synthetic peptide 136 Ser TyrVal Pro Ser Ala Glu Gln Ile 5 137 8 PRT Artificial Sequence syntheticpeptide 137 Phe Glu Gln Asn Thr Ala Gln Pro 5 138 8 PRT ArtificialSequence synthetic peptide 138 Phe Glu Gln Asn Thr Ala Gln Ala 5 139 9PRT Artificial Sequence synthetic peptide 139 Glu Ala Asp Pro Thr GlyHis Ser Tyr 5 140 9 PRT Artificial Sequence synthetic peptide 140 GluVal Asp Pro Ile Gly His Leu Tyr 5 141 9 PRT Artificial Sequencesynthetic peptide 141 Ala Ala Gly Ile Gly Ile Leu Thr Val 5 142 9 PRTArtificial Sequence synthetic peptide 142 Tyr Leu Glu Pro Gly Pro ValThr Ala 5 143 10 PRT Artificial Sequence synthetic peptide 143 Ile LeuAsp Gly Thr Ala Thr Leu Arg Leu 5 10 144 9 PRT Artificial Sequencesynthetic peptide 144 Met Leu Leu Ala Leu Leu Tyr Cys Leu 5 145 9 PRTArtificial Sequence synthetic peptide 145 Tyr Met Asn Gly Thr Met SerGln Val 5 146 9 PRT Artificial Sequence synthetic peptide 146 Leu ProTyr Leu Gly Trp Leu Val Phe 5 147 9 PRT Artificial Sequence syntheticpeptide 147 Phe Gly Pro Tyr Lys Leu Asn Arg Leu 5 148 8 PRT ArtificialSequence synthetic peptide 148 Lys Ser Pro Trp Phe Thr Thr Leu 5 149 10PRT Artificial Sequence synthetic peptide 149 Gly Pro Pro His Ser AsnAsn Phe Gly Tyr 5 10 150 9 PRT Artificial Sequence synthetic peptide 150Ile Ser Thr Gln Asn His Arg Ala Leu 5 151 10 PRT Artificial Sequencesynthetic peptide 151 Tyr Gly Ile Leu Gly Lys Val Phe Thr Leu 5 10 152 9PRT Artificial Sequence synthetic peptide 152 Ser Leu Tyr Asn Thr ValAla Thr Leu 5 153 8 PRT Artificial Sequence synthetic peptide 153 LeuPhe Trp Pro Phe Glu Trp Ile 5 154 8 PRT Artificial Sequence syntheticpeptide 154 Asp Gly Val Gly Ser Phe Ile Gly 5 155 8 PRT ArtificialSequence synthetic peptide 155 Glu Ser Leu Trp Asn Pro Gln Gly 5 156 8PRT Artificial Sequence synthetic peptide 156 Leu His Phe Asp Val LeuTrp Arg 5 157 8 PRT Artificial Sequence synthetic peptide 157 Cys HisLeu Lys Met Val Pro Trp 5 158 8 PRT Artificial Sequence syntheticpeptide 158 Asn Ser Val Leu Val Cys Glu Leu 5 159 8 PRT ArtificialSequence synthetic peptide 159 Asp Arg Gly His Ser Thr Tyr Ser 5 160 8PRT Artificial Sequence synthetic peptide 160 Asp Val Trp Gly Trp ValThr Trp 5 161 8 PRT Artificial Sequence synthetic peptide 161 Ile GlnPhe Arg Val Glu Leu Phe 5 162 8 PRT Artificial Sequence syntheticpeptide 162 Leu Trp Leu Glu Leu Ser Leu Ser 5 163 8 PRT ArtificialSequence synthetic peptide 163 Val Gly Ile Cys Ala Leu Gly Phe 5 164 8PRT Artificial Sequence synthetic peptide 164 Pro Tyr Pro Ser Gly LeuAsp Ser 5 165 8 PRT Artificial Sequence synthetic peptide 165 Phe TrpGly Val Leu Pro Tyr Pro 5 166 8 PRT Artificial Sequence syntheticpeptide 166 Phe Thr His Gly Ile Ser Leu Tyr 5 167 8 PRT ArtificialSequence synthetic peptide 167 Asn His Ser Phe Gly Gly Ser Thr 5 168 8PRT Artificial Sequence synthetic peptide 168 Val Asp Tyr Val Tyr PheHis His 5 169 8 PRT Artificial Sequence synthetic peptide 169 Phe LeuAsp Ile Ile Gly Tyr Gly 5 170 8 PRT Artificial Sequence syntheticpeptide 170 Trp Asp Asp Leu Leu His Gly Arg 5 171 8 PRT ArtificialSequence synthetic peptide 171 Leu Arg Leu Leu Gly Thr Leu Asn 5 172 8PRT Artificial Sequence synthetic peptide 172 Phe Glu Gln His Asn GlnGlu Pro 5 173 8 PRT Artificial Sequence synthetic peptide 173 Phe ValGly Thr Val Thr Trp Ser 5 174 8 PRT Artificial Sequence syntheticpeptide 174 Leu Trp Ala Leu Thr Tyr Arg Gly 5 175 8 PRT ArtificialSequence synthetic peptide 175 Ser Trp Gly Ser Asn Gly Gly Phe 5 176 8PRT Artificial Sequence synthetic peptide 176 Asp Met Trp Arg Arg AlaVal Gln 5 177 8 PRT Artificial Sequence synthetic peptide 177 Cys ArgVal Ile Tyr His Ala Thr 5 178 8 PRT Artificial Sequence syntheticpeptide 178 Met Val Val Ala Arg Cys Gly His 5 179 8 PRT ArtificialSequence synthetic peptide 179 His Met Trp Ile Asn Trp Val Gln 5 180 8PRT Artificial Sequence synthetic peptide 180 Cys Ala Gly Arg Cys PheGly Tyr 5 181 8 PRT Artificial Sequence synthetic peptide 181 Cys ThrHis Val Leu Ala Tyr Ser 5 182 8 PRT Artificial Sequence syntheticpeptide 182 Ser Trp Met Pro Trp Leu Thr Met 5 183 8 PRT ArtificialSequence synthetic peptide 183 Leu Glu Trp Cys Ile Trp Arg Tyr 5 184 8PRT Artificial Sequence synthetic peptide 184 Cys Leu Ala Cys Ile IleHis Ser 5 185 8 PRT Artificial Sequence synthetic peptide 185 Phe TrpPhe Pro Trp Asp Arg Ser 5 186 8 PRT Artificial Sequence syntheticpeptide 186 Trp Arg Thr Gly Val Phe His Gly 5 187 8 PRT ArtificialSequence synthetic peptide 187 Met His Leu Arg Val Ala Asp Arg 5 188 8PRT Artificial Sequence synthetic peptide 188 Ala Leu Asp Leu Tyr LeuTyr Val 5 189 8 PRT Artificial Sequence synthetic peptide 189 Phe PheTrp Phe Thr Leu Lys Glu 5 190 8 PRT Artificial Sequence syntheticpeptide 190 Leu Ser Phe Ala Gly Trp Gly Val 5 191 8 PRT ArtificialSequence synthetic peptide 191 Met Met Met Leu Gly Arg Ala Pro 5 192 8PRT Artificial Sequence synthetic peptide 192 Trp Ser Phe Tyr Thr TrpLeu Asn 5 193 8 PRT Artificial Sequence synthetic peptide 193 Phe ValTrp Met Arg Trp Ile Asp 5 194 8 PRT Artificial Sequence syntheticpeptide 194 Met Gln Val Asn Thr Pro Asp Asn 5 195 8 PRT ArtificialSequence synthetic peptide 195 Phe Trp Gly Trp Leu Ile Pro Trp 5 196 7PRT Artificial Sequence synthetic peptide 196 Trp Gly Trp Val Trp TrpAsp 5 197 8 PRT Artificial Sequence synthetic peptide 197 Trp Ile PhePro Trp Ile Gln Leu 5 198 8 PRT Artificial Sequence synthetic peptide198 Trp Met Phe Asn Trp Pro Trp Tyr 5 199 8 PRT Artificial Sequencesynthetic peptide 199 Met Asn Met Ile Val Leu Asp Lys 5 200 8 PRTArtificial Sequence synthetic peptide 200 Phe Trp Gly Trp Pro Gly TrpSer 5 201 8 PRT Artificial Sequence synthetic peptide 201 Trp Leu IleArg Val Gly Thr Ala 5 202 8 PRT Artificial Sequence synthetic peptide202 Gly Leu Leu Thr His Leu Ile Trp 5 203 8 PRT Artificial Sequencesynthetic peptide 203 Leu Trp Trp Leu Asn Val His Gly 5 204 8 PRTArtificial Sequence synthetic peptide 204 Trp Trp Trp Ile Asn Asp GluSer 5 205 8 PRT Artificial Sequence synthetic peptide 205 Ala Asn ProSer Leu Ala Thr Tyr 5 206 8 PRT Artificial Sequence synthetic peptide206 Trp Leu Gln Gly Trp Trp Gly Trp 5 207 8 PRT Artificial Sequencesynthetic peptide 207 Met Met Pro Val Thr Ser Phe Arg 5 208 8 PRTArtificial Sequence synthetic peptide 208 Gly Trp Met Asp Trp Trp TyrTyr 5 209 8 PRT Artificial Sequence synthetic peptide 209 Leu Ala SerMet Arg Asn Ser Met 5 210 8 PRT Artificial Sequence synthetic peptide210 Asp Leu Met Arg Trp Leu Gly Leu 5 211 8 PRT Artificial Sequencesynthetic peptide 211 Tyr Phe Tyr Ala Trp Trp Leu Asp 5 212 8 PRTArtificial Sequence synthetic peptide 212 Leu Gly His Leu Trp Thr GlnVal 5 213 8 PRT Artificial Sequence synthetic peptide 213 Leu Trp TrpArg Asp Val Met Ala 5 214 8 PRT Artificial Sequence synthetic peptide214 Phe Ile Trp Trp Ala Pro Leu Ala 5 215 8 PRT Artificial Sequencesynthetic peptide 215 Gly Ser Val Gly Gly Gly Val Val 5 216 8 PRTArtificial Sequence synthetic peptide 216 Asp Ser His Asp Asp Trp ArgMet 5 217 8 PRT Artificial Sequence synthetic peptide 217 Phe Trp ArgPhe Asp Tyr Tyr Phe 5 218 8 PRT Artificial Sequence synthetic peptide218 Trp Thr Trp Trp Glu Trp Leu Ala 5 219 8 PRT Artificial Sequencesynthetic peptide 219 Trp Leu Trp Asp Trp Ile Val Val 5 220 8 PRTArtificial Sequence synthetic peptide 220 Gly Trp Thr Trp Phe Phe AspMet 5 221 8 PRT Artificial Sequence synthetic peptide 221 Ala Trp TrpGln His Phe Ile Val 5 222 8 PRT Artificial Sequence synthetic peptide222 Leu Trp Trp Asp Ile Ile Thr Gly 5 223 8 PRT Artificial Sequencesynthetic peptide 223 Phe Thr Tyr Gly Ser Arg Trp Leu 5 224 8 PRTArtificial Sequence synthetic peptide 224 Phe Ser Leu Trp Pro Leu AlaTrp 5 225 8 PRT Artificial Sequence synthetic peptide 225 Gly Ile IleLeu Gly Tyr Asn Val 5 226 8 PRT Artificial Sequence synthetic peptide226 Ser Trp Met Thr Trp Ile Glu His 5 227 8 PRT Artificial Sequencesynthetic peptide 227 Gly Trp Trp Val Thr Trp Pro Trp 5 228 8 PRTArtificial Sequence synthetic peptide 228 Val Val Ser Pro Trp Trp LeuGly 5 229 8 PRT Artificial Sequence synthetic peptide 229 Asn Val LeuSer Arg Gly Phe Ser 5 230 8 PRT Artificial Sequence synthetic peptide230 Ser Phe Glu Ser Leu Gly Gly Leu 5 231 8 PRT Artificial Sequencesynthetic peptide 231 Ile Thr Lys Gly Ser Ser Phe Pro 5 232 8 PRTArtificial Sequence synthetic peptide 232 Leu Asp Trp Ala Arg Lys LeuArg 5 233 8 PRT Artificial Sequence synthetic peptide 233 Thr Ala TrpAsn Leu Leu Gly Tyr 5 234 8 PRT Artificial Sequence synthetic peptide234 Phe Gly Gln Gly Ile Lys His Val 5 235 8 PRT Artificial Sequencesynthetic peptide 235 Asp Val Val Trp Gln Arg Leu Leu 5 236 8 PRTArtificial Sequence synthetic peptide 236 Tyr Val Asp Arg Phe Ile GlyTrp 5 237 8 PRT Artificial Sequence synthetic peptide 237 Lys Met AlaArg Pro Glu Gly Asn 5 238 8 PRT Artificial Sequence synthetic peptide238 Leu Gly Arg Trp Gly His Glu Ser 5 239 8 PRT Artificial Sequencesynthetic peptide 239 Ser Ile Trp Ser Leu Leu Val Leu 5 240 8 PRTArtificial Sequence synthetic peptide 240 Val Trp Leu Asp Leu Leu LeuSer 5 241 8 PRT Artificial Sequence synthetic peptide 241 Tyr Leu AspThr Ser Leu Phe Gly 5 242 8 PRT Artificial Sequence synthetic peptide242 Thr Trp Trp Pro Ser Ile Thr Trp 5 243 8 PRT Artificial Sequencesynthetic peptide 243 Tyr Gly Leu Trp Trp Phe Pro Trp 5 244 8 PRTArtificial Sequence synthetic peptide 244 Phe Ser Pro Ala Asp Thr ArgTyr 5 245 8 PRT Artificial Sequence synthetic peptide 245 Cys Asn ArgLeu Gln Ile Asp Cys 5 246 8 PRT Artificial Sequence synthetic peptide246 Ser Leu Val Ala Ala Arg Asn Leu 5 247 8 PRT Artificial Sequencesynthetic peptide 247 Phe Thr Ile His Asn Val Ala Val 5 248 8 PRTArtificial Sequence synthetic peptide 248 Met Gly Pro Leu Gly Pro LeuLeu 5 249 8 PRT Artificial Sequence synthetic peptide 249 Arg Gln LeuSer Glu Leu Phe Val 5 250 8 PRT Artificial Sequence synthetic peptide250 Arg Val Val Cys Gln Ala Leu Leu 5 251 8 PRT Artificial Sequencesynthetic peptide 251 Trp Pro His Leu Trp Trp Leu Asp 5 252 8 PRTArtificial Sequence synthetic peptide 252 Trp Met Asp Trp Val Trp HisThr 5 253 8 PRT Artificial Sequence synthetic peptide 253 Trp Trp GlyTyr Leu Ile Cys Gln 5 254 8 PRT Artificial Sequence synthetic peptide254 Phe Arg Gly Leu Ser Glu Gly Pro 5 255 8 PRT Artificial Sequencesynthetic peptide 255 Ser Trp Phe Asp Trp Leu Val Ala 5 256 8 PRTArtificial Sequence synthetic peptide 256 Val Val Met Trp Tyr Ser ValAsp 5 257 7 PRT Artificial Sequence synthetic peptide 257 Trp Gly TrpSer Leu Ala Thr 5 258 8 PRT Artificial Sequence synthetic peptide 258Leu Gly Trp Phe Asp Arg Phe Phe 5 259 8 PRT Artificial Sequencesynthetic peptide 259 Ala Trp Trp Trp Pro Thr Tyr Val 5 260 8 PRTArtificial Sequence synthetic peptide 260 Gly Phe Leu Ser Ser Trp PheLeu 5 261 8 PRT Artificial Sequence synthetic peptide 261 Gly Val IleAsn Cys Ala Gly Thr 5 262 8 PRT Artificial Sequence synthetic peptide262 Val Cys Ala Arg Ala Ala His Leu 5 263 8 PRT Artificial Sequencesynthetic peptide 263 Gly Asn Ser Tyr Gly Asp Gly Gly 5 264 8 PRTArtificial Sequence synthetic peptide 264 Gly Phe Leu Ser Ser Trp PheLeu 5 265 8 PRT Artificial Sequence synthetic peptide 265 Phe Asp GlnPro Gly Arg Phe Leu 5 266 8 PRT Artificial Sequence synthetic peptide266 Arg Ser His Ala Thr Gly Val Val 5 267 8 PRT Artificial Sequencesynthetic peptide 267 Gly Tyr Trp Ala Met Met Ser Trp 5 268 8 PRTArtificial Sequence synthetic peptide 268 Cys His Ser Met Trp Asp GlyLeu 5 269 8 PRT Artificial Sequence synthetic peptide 269 Phe Ile TrpArg Gly Trp Pro His 5 270 8 PRT Artificial Sequence synthetic peptide270 Leu Ser Phe Leu Gly Gly Arg Leu 5 271 8 PRT Artificial Sequencesynthetic peptide 271 Phe Ser Gly Val Arg Gln Pro Asn 5 272 8 PRTArtificial Sequence synthetic peptide 272 Trp Gly Trp Met Pro Phe TyrTyr 5 273 8 PRT Artificial Sequence synthetic peptide 273 Phe Thr ArgPro Ala Val Val Asp 5 274 8 PRT Artificial Sequence synthetic peptide274 Asp Leu Trp Thr Trp Leu Gly Leu 5 275 8 PRT Artificial Sequencesynthetic peptide 275 Cys Asp Thr Ala Ala Val Ala Asp 5 276 8 PRTArtificial Sequence synthetic peptide 276 Trp Trp Val Lys His His MetLeu 5 277 8 PRT Artificial Sequence synthetic peptide 277 Ile Ala PheLeu Arg Asp Asn Arg 5 278 8 PRT Artificial Sequence synthetic peptide278 Leu Ala Arg Pro Asp His Tyr Ser 5 279 8 PRT Artificial Sequencesynthetic peptide 279 Met Glu Ser Lys Arg Trp Thr Val 5 280 8 PRTArtificial Sequence synthetic peptide 280 Met Ile Leu Lys Gly Tyr SerArg 5 281 8 PRT Artificial Sequence synthetic peptide 281 Ala Pro SerAsp Tyr Asp Glu Ser 5 282 8 PRT Artificial Sequence synthetic peptide282 His Trp Leu Arg Ser Lys Arg Thr 5 283 8 PRT Artificial Sequencesynthetic peptide 283 Gly Ala Arg Val Trp Asn Tyr Gln 5 284 8 PRTArtificial Sequence synthetic peptide 284 Leu Ser Asn Trp Asn Met ArgLeu 5 285 8 PRT Artificial Sequence synthetic peptide 285 Cys Gly AlaAla Gln Gln Gly Met 5 286 8 PRT Artificial Sequence synthetic peptide286 Gly Ser Ser Met Val Val Gln Arg 5 287 15 PRT Artificial Sequencesynthetic peptide 287 Lys Arg Gln Ile Tyr Thr Asp Leu Glu Met Asn ArgLeu Gly Lys 5 10 15 288 15 PRT Artificial Sequence synthetic peptide 288Leu Ser Ser Leu Phe Arg Pro Lys Arg Arg Pro Ile Tyr Lys Ser 5 10 15 28913 PRT Artificial Sequence synthetic peptide 289 Lys Leu Ile Gly Val LeuSer Ser Leu Phe Arg Pro Lys 5 10 290 15 PRT Artificial Sequencesynthetic peptide 290 Arg Arg Pro Ile Tyr Lys Ser Asp Val Gly Met AlaHis Phe Arg 5 10 15 291 11 PRT Artificial Sequence synthetic peptide 291Cys Lys Ile Gln Ser Thr Pro Val Lys Gln Ser 5 10 292 15 PRT ArtificialSequence synthetic peptide 292 Glu Gly Met Ile Asp Gly Trp Tyr Gly PheArg His Gln Asn Cys 5 10 15 293 11 PRT Artificial Sequence syntheticpeptide 293 Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser Cys 5 10 294 10 PRTArtificial Sequence synthetic peptide 294 Ser Asn Gly Ser Leu Gln CysArg Ile Cys 5 10 295 6 PRT Artificial Sequence synthetic peptide 295 GlyLys Trp Val Tyr Ile 5 296 6 PRT Artificial Sequence synthetic peptide296 Ala Lys Arg Glu Thr Lys 5 297 6 PRT Artificial Sequence syntheticpeptide 297 Lys Trp Val His Leu Phe 5 298 6 PRT Artificial Sequencesynthetic peptide 298 Arg Leu Val Leu Val Leu 5 299 6 PRT ArtificialSequence synthetic peptide 299 Trp Lys Trp Gly Ile Tyr 5 300 6 PRTArtificial Sequence synthetic peptide 300 Ser Ser His Ala Ser Ala 5 3016 PRT Artificial Sequence synthetic peptide 301 Trp Gly Pro Trp Ser Phe5 302 6 PRT Artificial Sequence synthetic peptide 302 Ala Ile Pro GlyLys Val 5 303 6 PRT Artificial Sequence synthetic peptide 303 Arg ValHis Asp Pro Ala 5 304 6 PRT Artificial Sequence synthetic peptide 304Arg Ser Val Ser Ser Phe 5 305 6 PRT Artificial Sequence syntheticpeptide 305 Leu Gly Thr Arg Lys Gly 5 306 6 PRT Artificial Sequencesynthetic peptide 306 Lys Asp Pro Leu Phe Asn 5 307 6 PRT ArtificialSequence synthetic peptide 307 Leu Ser Gln His Thr Asn 5 308 6 PRTArtificial Sequence synthetic peptide 308 Asn Arg Leu Leu Leu Thr 5 3096 PRT Artificial Sequence synthetic peptide 309 Tyr Pro Leu Trp Val Ile5 310 6 PRT Artificial Sequence synthetic peptide 310 Leu Leu Ile IleAsp Arg 5 311 6 PRT Artificial Sequence synthetic peptide 311 Arg ValIle Ser Leu Gln 5 312 6 PRT Artificial Sequence synthetic peptide 312Glu Val Ser Arg Glu Asp 313 6 PRT Artificial Sequence synthetic peptide313 Ser Ile Leu Arg Ser Thr 5 314 6 PRT Artificial Sequence syntheticpeptide 314 Pro Gly Leu Val Trp Leu 5 315 6 PRT Artificial Sequencesynthetic peptide 315 Val Lys Lys Leu Tyr Ile 5 316 6 PRT ArtificialSequence synthetic peptide 316 Asn Asn Arg Leu Leu Asp 5 317 6 PRTArtificial Sequence synthetic peptide 317 Ser Lys Gly Arg Trp Gly 5 3186 PRT Artificial Sequence synthetic peptide 318 Ile Arg Pro Ser Gly Ile5 319 6 PRT Artificial Sequence synthetic peptide 319 Ala Ser Leu CysPro Thr 5 320 6 PRT Artificial Sequence synthetic peptide 320 Asp ValPro Gly Leu Arg 5 321 6 PRT Artificial Sequence synthetic peptide 321Arg His Arg Glu Val Gln 5 322 6 PRT Artificial Sequence syntheticpeptide 322 Leu Ala Arg Lys Arg Ser 5 323 6 PRT Artificial Sequencesynthetic peptide 323 Ser Val Leu Asp His Val 5 324 6 PRT ArtificialSequence synthetic peptide 324 Asn Leu Leu Arg Arg Ala 5 325 6 PRTArtificial Sequence synthetic peptide 325 Ser Gly Ile Ser Ala Trp 5 3266 PRT Artificial Sequence synthetic peptide 326 Phe Tyr Pro Trp Val Arg5 327 6 PRT Artificial Sequence synthetic peptide 327 Lys Leu Phe LeuPro Leu 5 328 6 PRT Artificial Sequence synthetic peptide 328 Thr ProThr Leu Ser Asp 5 329 6 PRT Artificial Sequence synthetic peptide 329Thr His Ser Leu Ile Leu 5 330 6 PRT Artificial Sequence syntheticpeptide 330 Leu Leu Leu Leu Ser Arg 5 331 6 PRT Artificial Sequencesynthetic peptide 331 Leu Leu Arg Val Arg Ser 5 332 6 PRT ArtificialSequence synthetic peptide 332 Glu Arg Arg Ser Arg Gly 5 333 6 PRTArtificial Sequence synthetic peptide 333 Arg Met Leu Gln Leu Ala 5 3346 PRT Artificial Sequence synthetic peptide 334 Arg Gly Trp Ala Asn Ser5 335 6 PRT Artificial Sequence synthetic peptide 335 Arg Pro Phe TyrSer Tyr 5 336 6 PRT Artificial Sequence synthetic peptide 336 Ser SerSer Trp Asn Ala 5 337 6 PRT Artificial Sequence synthetic peptide 337Leu Gly His Leu Glu Glu 5 338 6 PRT Artificial Sequence syntheticpeptide 338 Ser Ala Val Thr Asn Thr 5 339 6 PRT Artificial Sequencesynthetic peptide 339 Leu Arg Arg Ala Ser Leu 5 340 6 PRT ArtificialSequence synthetic peptide 340 Leu Arg Arg Trp Ser Leu 5 341 6 PRTArtificial Sequence synthetic peptide 341 Lys Trp Val His Leu Phe 5 3426 PRT Artificial Sequence synthetic peptide 342 Asn Arg Leu Leu Leu Thr5 343 6 PRT Artificial Sequence synthetic peptide 343 Ala Arg Leu LeuLeu Thr 5 344 6 PRT Artificial Sequence synthetic peptide 344 Asn AlaLeu Leu Leu Thr 5 345 6 PRT Artificial Sequence synthetic peptide 345Asn Arg Leu Ala Leu Thr 5 346 6 PRT Artificial Sequence syntheticpeptide 346 Asn Leu Leu Arg Leu Thr 5 347 6 PRT Artificial Sequencesynthetic peptide 347 Asn Arg Leu Trp Leu Thr 5 348 6 PRT ArtificialSequence synthetic peptide 348 Asn Arg Leu Leu Leu Ala 5 349 12 PRTArtificial Sequence synthetic peptide 349 Met Gln Glu Arg Ile Thr LeuLys Asp Tyr Ala Met 5 10

We claim:
 1. A composition for inducing a therapeutic immune response ina subject, comprising: (a) a target antigen; and (b) a heat shockprotein; wherein the target antigen and the heat shock protein arecombined in vitro under conditions wherein binding of target antigen toheat shock protein occurs to form a target antigen/heat shock proteincomplex; wherein the administration of the target antigen/heat shockprotein complex to the subject induces an immune response comprising acytotoxic cellular component.
 2. The composition of claim 1, wherein theheat shock protein is hsp70.
 3. The composition of claim 1, wherein theheat shock protein is gp96.
 4. The composition of claim 1, wherein theheat shock protein is hsp40.
 5. The composition of claim 1, wherein theheat shock protein is BiP.
 6. The composition of any of claims 1 to 5,wherein the target antigen is a hybrid antigen.
 7. The compositionaccording to claim 6 wherein the hybrid antigen comprises an antigenicdomain derived from a first source and a binding domain which binds to aheat shock protein from a second source different from the first source.8. The composition of claim 7, wherein the binding domain comprises atleast a heptameric region having the sequence HyXHyXHyXHy where Hyrepresents a hydrophobic amino acid residue and X is any amino acid. 9.The composition of claim 7, wherein the binding domain comprises aregion having the sequence His Trp Asp Phe Ala Trp Pro Trp [Seq. ID No.1].
 10. A composition for inducing a therapeutic immune response in asubject, comprising: (a) a nucleic acid molecule comprising a regionencoding a target antigen operably linked to a promoter element; and (b)a nucleic acid molecule comprising a region encoding a heat shockprotein operably linked to a promoter element; wherein the introductionof the nucleic acids of (a) and (b) into a cell result in the binding oftarget antigen to heat shock protein.
 11. The composition of claim 10,wherein the nucleic acid molecules of (a) and (b) are comprised in thesame vector.
 12. The composition of claim 10 or 11, wherein the heatshock protein is hsp70.
 13. The composition of claim 10 or 11, whereinthe heat shock protein is gp96.
 14. The composition of claim 10 or 11,wherein the heat shock protein is hsp40.
 15. The composition of claim 10or 11, wherein the heat shock protein is BiP.
 16. The composition of anyof claims 10 to 15, wherein the target antigen is a hybrid antigen. 17.The composition according to claim 16, wherein the hybrid antigencomprises an antigenic domain derived from a first source and a bindingdomain which binds to a heat shock protein from a second sourcedifferent from the first source.
 18. The composition of claim 17,wherein the binding domain comprises at least a heptameric region havingthe sequence HyXHyXHyXHy where Hy represents a hydrophobic amino acidresidue and X is any amino acid.
 19. The composition of claim 17,wherein the binding domain comprises a region having the sequence HisTrp Asp Phe Ala Trp Pro Trp [Seq. ID No. 1].
 20. A method of inducing animmune response in a subject in need of such treatment, comprisingadministering to the subject a therapeutically effective amount of thecomposition of any of claims 1 to
 19. 21. A hybrid peptide comprising:(a) an antigenic domain derived from a first source; and (b) a bindingdomain which binds to a heat shock protein, said binding domain beingderived from a second source different from the first source.
 22. Thehybrid peptide of claim 21, wherein the antigenic domain is derived froma virus, a parasite, a mycoplasma, a fungus or a bacterium.
 23. Thehybrid peptide of any of claims 21-22, wherein the antigenic domainelicits an immune response to a neoplastic disease.
 24. The hybridpeptide of claim 23, wherein the neoplastic disease is selected fromamong a sarcoma, a lymphoma, a carcinoma, a leukemia and a melanoma. 25.The hybrid peptide of any of claims 21 to 24, wherein the binding domaincomprises at least a heptameric region having the sequence HyXHyXHyXHywhere Hy represents a hydrophobic amino acid residue and X is any aminoacid.
 26. The hybrid peptide of any of claim 21 to 24, wherein thebinding domain comprises a region having the sequence His Trp Asp PheAla Trp Pro Trp [Seq. ID No. 1].
 27. The hybrid peptide of any of claims21 to 24, wherein the binding domain comprises at least a pentapeptideregion selected from among Gln Lys Arg Ala Ala, and [Seq. ID No. 5] ArgArg Arg Ala Ala. [Seq. ID No. 6]


28. A polynucleotide construct comprising: (a) a region encoding ahybrid peptide comprising an antigenic domain derived from a firstsource; and a binding domain which binds to a heat shock protein saidbinding domain being derived from a second source different from thefirst source; (b) a promoter effective to promote expression on thehybrid peptide in mammalian cells.
 29. The polynucleotide construct ofclaim 28, wherein the antigenic domain is derived from a virus, aparasite, a mycoplasma, a fungus or a bacterium.
 30. The polynucleotideconstruct of claim 28, wherein the antigenic domain elicits an immuneresponse to a neoplastic disease.
 31. The polynucleotide construct ofclaim 30, wherein the neoplastic disease is selected from among asarcoma, a lymphoma, a carcinoma, a leukemia and a melanoma.
 32. Thepolynucleotide construct of any of claims 28 to 31, wherein the bindingdomain comprises at least a heptameric region having the sequenceHyXHyXHyXHy where Hy represents a hydrophobic amino acid residue and Xis any amino acid.
 33. The polynucleotide construct of any of claims 28to 31, wherein the binding domain comprises a region having the sequenceHis Trp Asp Phe Ala Trp Pro Trp [Seq. ID No. 1].
 34. The polynucleotideconstruct of any of claims 28 to 31, wherein the binding domaincomprises at least a pentapeptide region selected from among Gln Lys ArgAla Ala, and [Seq. ID No. 5] Arg Arg Arg Ala Ala. [Seq. ID No. 6]